U.S. patent number 5,334,505 [Application Number 07/633,231] was granted by the patent office on 1994-08-02 for n- and o-substituted aminophenols, method and use for diagnosis.
This patent grant is currently assigned to Boehringer Mannheim GmbH. Invention is credited to Dieter Mangold, Gerd Zimmermann.
United States Patent |
5,334,505 |
Zimmermann , et al. |
August 2, 1994 |
**Please see images for:
( Certificate of Correction ) ** |
N- and O-substituted aminophenols, method and use for diagnosis
Abstract
The present invention provides N- and O-substituted aminophenol
derivatives of the general formula ##STR1## wherein R.sup.1,
R.sup.2, R.sup.3, G and L are as hereinbefore defined. The present
invention also provides intermediates for the preparation of these
aminophenol derivatives of general formula (I), as well as the use
of the aminophenol derivatives of general formula (I) for the
determination of hydrolyses, as well as for the preparation of
agents for carrying out determinations of hydrolyses.
Inventors: |
Zimmermann; Gerd (Mannheim,
DE), Mangold; Dieter (Maxdorf, DE) |
Assignee: |
Boehringer Mannheim GmbH
(Mannheim, DE)
|
Family
ID: |
6396066 |
Appl.
No.: |
07/633,231 |
Filed: |
December 21, 1990 |
Foreign Application Priority Data
|
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|
|
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Dec 21, 1989 [DE] |
|
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3942355 |
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Current U.S.
Class: |
435/18; 435/23;
435/810; 536/118; 536/120; 536/17.3 |
Current CPC
Class: |
C07D
471/04 (20130101); C07D 487/04 (20130101); C07D
513/04 (20130101); C07F 9/6561 (20130101); C07H
15/203 (20130101); C07H 15/26 (20130101); C12Q
1/34 (20130101); C12Q 2334/00 (20130101); Y10S
435/81 (20130101) |
Current International
Class: |
C07D
513/00 (20060101); C07D 513/04 (20060101); C07D
471/00 (20060101); C07F 9/00 (20060101); C07D
471/04 (20060101); C07D 487/00 (20060101); C07H
15/00 (20060101); C07H 15/26 (20060101); C07F
9/6561 (20060101); C07H 15/203 (20060101); C07D
487/04 (20060101); C12Q 1/34 (20060101); C07H
017/00 (); G01N 031/14 () |
Field of
Search: |
;435/18,810,7,23
;536/17.3,118,120 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Wityshyn; Michael G.
Assistant Examiner: Leary; Louise N.
Attorney, Agent or Firm: Felfe & Lynch
Claims
We claim:
1. A process for colorimetric determination of a hydrolase enzyme
by reaction of a chromogenic enzyme substrate with the enzyme,
oxidation of a leuko colored material liberated from the
chromogenic enzyme substrate by the enzyme and determination of the
resulting colored material as a measure for the amount of enzyme,
comprising
reacting the hydrolase enzyme with an N- and O-substituted
aminophenyl derivative as the chromogenic enzyme substrate of the
formula: ##STR99## wherein G is the residue of an organic or
inorganic acid or a glycoside residue,
R.sup.1 and R.sup.2, which are the same or different, are hydrogen,
halogen, SO.sub.3 H, PO.sub.3 H.sub.2 or a salt of the acid
residues of SO.sub.3 H or PO.sub.3 H.sub.2, hydroxyl, nitro,
carboxyl, carboxamido, cyano, alkyl, alkenyl, alkoxy,
alkylsulphinyl, alkylsulphonyl, alkoxycarbonyl, alkylcarbonyl, aryl
or aralkyl unsubstituted or substituted at least once by hydroxyl,
carboxyl, halogen, cyano, SO.sub.3 H, PO.sub.3 H.sub.2 or a salt of
one of the acid residues of SO.sub.3 H or PO.sub.3 H.sub.2 or,
when two substituents of R.sup.1 and R.sup.2 are present on
neighboring carbon atoms, R.sup.1 and R.sup.2 together represent a
1,4-butadiendiyl radical which is unsubstituted or substituted at
least once by SO.sub.3 H, PO.sub.3 H.sub.2 or a salt of the acid
groups of SO.sub.3 H or PO.sub.3 H.sub.2, an alkyl or a carboxyl
group,
R.sup.3 is hydrogen, --CO--COOH, SO.sub.3 H, PO.sub.3 H.sub.2 or a
salt of the acid groups of SO.sub.3 H or PO.sub.3 H.sub.2, an
alkylcarbonyl radical unsubstituted or substituted at least once by
halogen, COOH, SO.sub.3 H or PO.sub.3 H.sub.2 or a salt of the acid
groups of SO.sub.3 H or PO.sub.3 H.sub.2 or an arylcarbonyl radical
unsubstituted or substituted at least once by SO.sub.3 H, PO.sub.3
H.sub.2 or a salt of the acid groups of SO.sub.3 H or PO.sub.3
H.sub.2, and
L is a radical of the formula: ##STR100## wherein R.sup.4 and
R.sup.5, which are the same or different, are alkyl or together
represent a saturated 3-6 member hydrocarbon chain which is
uninterrupted or interrupted by oxygen, sulphur or nitrogen and
wherein alkyl or the hydrocarbon chain is unsubstituted or
substituted at least once by hydroxyl, carboxyl, alkoxycarbonyl,
alkoxy, SO.sub.3 H, PO.sub.3 H.sub.2 or a salt of one of the acid
groups of SO.sub.3 H or PO.sub.3 H.sub.2 or halogen, and
R.sup.6 and R.sup.7, which can be the same or different, are
hydrogen, halogen, hydroxyl, carboxamido, an alkyl, alkoxy,
alkylcarbonyl, alkoxycarbonyl, aryl or aralkyl radical
unsubstituted or substituted at least once by hydroxyl, carboxyl,
halogen, SO.sub.3 H, PO.sub.3 H.sub.2 or a salt of one of the acid
groups of SO.sub.3 H or PO.sub.3 H.sub.2, or
L is a pyrazolo-heterocylic radical of the formula: ##STR101##
wherein X-Y signifies NR.sup.8 --CO or N.dbd.CR.sup.9, wherein
R.sup.8 is hydrogen or an alkyl and R.sup.9 is alkyl, alkenyl,
alkoxy, alkylthio, aryl, aralkyl, in each case unsubstituted or
substituted by hydroxyl, dialkylphosphinyl, carboxyl, SO.sub.3 H,
PO.sub.3 H.sub.2 or a salt of one of the acid groups of SO.sub.3 H
or PO.sub.3 H.sub.2, alkoxycarbonyl, amino, wherein the amino is
unsubstituted or substituted by one or two alkyl radicals which one
or two alkyl radicals are unsubstituted or substituted by at least
one hydroxyl, carboxyl or alkoxycarbonyl radical and wherein when
amino is substituted by two alkyl radicals, the alkyl radicals can
be joined to form a ring which, apart from the first nitrogen atom
of the amino group, is uninterrupted or is interrupted by oxygen,
sulphur or a second nitrogen atom, or the amino is unsubstituted or
substituted by one or two acyl radicals, alkoxy- or
aralkoxycarbonyl radicals, H.sub.2 N--CO, alkyl-, aralkyl- or
arylcarbamoyl radicals; or R.sup.9 is hydrogen, carboxyl,
alkoxycarbonyl, carboxamido or halogen, and
Z signifies NR.sup.10 --N.dbd.N, wherein R.sup.10 is hydrogen or an
alkyl or aralkyl radical, or
Z is an unsaturated 3-5 member chain of nitrogen atoms or of carbon
atoms, or said chain having at least one nitrogen or sulphur atom,
whereby carbon atoms in the chain are unsubstituted or substituted
by alkyl, alkoxy, hydroxyalkyl, alkylthio, hydroxyl, aralkyl, aryl,
carboxyl, carboxamido, alkoxycarbonyl, cyano, amino, wherein the
amino is unsubstituted or substituted by one or two alkyl radicals
which one or two alkyl radicals are unsubstituted or substituted by
at least one hydroxyl, carboxyl, alkoxycarbonyl, halogen, or
nitrogen which is not attached via a double bond and nitrogen is
unsubstituted or substituted by alkyl or aralkyl or two neighboring
chain substituents form an alkylene which alkylene is unsubstituted
or substituted or anellated with aryl and tautomers thereof.
2. The process of claim 1 for the determination of the enzyme
N-acetyl-.beta.-D-glucosaminidase, comprising using the chromogenic
substrate of claim 1 wherein G is an
N-acetyl-.beta.-D-glucosaminidyl radical substrate for the enzyme
N-acetyl-.beta.-D-glucosaminidase.
3. The process of claim 1 wherein G is a galactoside, glucoside,
mannoside, N-acetylglucosaminide or an oligosaccharide radical
consisting of 2 to 10 monosaccharide units.
4. The process of claim 1 wherein G is an
N-acetyl-.beta.-D-glucosaminidyl radical.
5. The process of claim 1 wherein G is PO.sub.3 MM', SO.sub.3 M, a
carboxyl-bound alkanecarboxylic acid, amino acid or oligopeptide
residue and M and M' are hydrogen, alkali metal, alkaline earth
metal or ammonium ions.
6. The process of claim 1 wherein akanecarboxylic acid residues of
G are acetic, propionic, butyric, palmitic, stearic, oleic,
linoleic or linolenic; and amino acid residues of G are glycine,
alanine, valine, leucine, isoleucine, phenylalanine or tyrosine and
a glycoside radical of G is .beta.-glucosidically bound
N-acetyl-2-D-glucosamine.
7. The process of claim 1 wherein alkyl is methyl, ethyl, propyl,
isobutyl, or tert.-butyl, the twice-substituted amino is
morpholino, hydroxyalkyl is 2-hydroxyethyl, 1-hydroxyethyl or
hydroxymethyl, aryl is phenyl, aralkyl is benzyl, araalkoxy is
benzyloxy, alkenyl is vinyl or allyl, halogen is fluorine or
chlorine, acyl is acetyl, phenylacetyl or benzoyl, alkylene is
butadiendiyl, diakylphosphinyl is dimethylphosphinyl and L is a
pyrazolo-heterocyclic racidal of formula III wherein X-Y is
N--CR.sup.9 wherein R.sup.9 is hydrogen or alkoxy.
8. The process of claim 1 wherein L is selected from the group
consisting of formulas (XIII) to XXIII and XXIV as follows:
##STR102## and tautomers thereof.
9. The process of claim 8 wherein L is selected from the group
consisting of formulas XIII, XIV, XV, XVII, XVIII, XX and tautomers
thereof.
10. The process of claim 1, wherein the chromogenic enzyme
substrate is the
.beta.-glucosidically-bound-N-acetyl-2-D-glucosaminide of
4-hydroxyphenyl-2-methylpyrazolo-pyridine-3-ylamine or of
4-hydroxyphenyl-pyrazolo-pyridine-3-ylamine.
11. The process of claim 1 wherein a stabilizing agent for the
chromogenic enzyme substrate is a 1-arylsemicarbazide of
formula:
wherein Ar is an aryl radical unsubstituted or substituted by
alkyl, alkoxy or halogen.
12. The process of claim 11 wherein the compound of formula XII is
selected from the group consisting of o-methyl, p-methyl,
o-methoxy, m-methoxy-, p-methoxy, o-chloro-m-chloro, m-methyl- and
unsubstituted phenyl semicarbazides and naphthyl semicarbazide.
13. The process of claim 1 wherein the chromogenic enzyme substrate
is in carrier-bound form.
14. The process of claim 11 wherein the chromogenic enzyme
substrate and the stabilizing agent are in carrier-bound form.
15. The process of claim 1 with the proviso that when G is an
alkanecarboxylic acid radical, R.sup.3 is hydrogen, an arylcarbonyl
radical or an alkylcarbonyl radical unsubstituted or substituted at
least once by halogen and L is a pyrazoloheterocyclic radical of
formula (III), wherein X-Y signifies N.dbd.CR.sup.9, whereby
R.sup.9 has the above-given meaning, then Z does not form a
1,2,3-triazole ring in which a nitrogen atom not connected via
double bond is substituted with hydrogen.
Description
The present invention is concerned with new N- and O-substituted
aminophenol derivatives and with a process for the preparation
thereof.
The present invention is also concerned with intermediates for the
preparation of the N- and O-substituted aminophenol derivatives
according to the present invention, as well as with processes for
the preparation thereof.
Furthermore, the present invention is concerned with the use of N-
and O-substituted aminophenol derivatives as hydrolase
substrates.
In addition, the present invention is concerned with a process for
the colorimetric determination of a hydrolase by reaction of a
chromogenic enzyme substrate with an enzyme, oxidation of a leuko
coloured material liberated by the enzyme from the substrate and
determination of the resulting colour material as a measure of the
amount of the enzyme.
The present invention is also concerned with a diagnostic agent for
the determination of a hydrolase, containing a chromogenic enzyme
substrate and an appropriate buffer substance.
Finally, the present invention is concerned with the use of an N-
or O-substituted aminophenol derivative for the preparation of a
diagnostic agent for the determination of a hydrolase.
By hydrolases are, in general, understood enzymes which
hydrolytically cleave bonds with the consumption of water. In
recent years, in clinical chemistry and in diagnosis, the
determination of the activity of such hydrolases has achieved
importance which cleave glycosidic and ether bonds. Furthermore, to
be here mentioned by way of example are esterases, for example the
enzymes cleaving carboxylic esters occurring in leukocytes or
phosphates, for example alkaline and acidic phosphates, which
hydrolyse phosphoric acid esters. For the diagnosis of diseases of
the kidneys and of the urogenital tract, it has proved to be useful
to detect leukocytes in urine on the basis of their inherent
esterolytic activity. The activity determination of acidic
phosphatase is a valuable means for the early diagnosis of
prostatic carcinoma. Alkaline phosphatase can be used as a
labelling enzyme for enzyme immunoassays.
Glycosidases, for example galactosidases, glucosidases,
mannosidases, amylase and N-acetyl-.beta.-D-glucosaminidase cleave
glycosidic bonds. In the human and animal organism, they fulfil a
plurality of physiological functions. Thus, for example,
.beta.-D-galactosidase plays an important part in the metabolism of
carbohydrates since, due to it, the hydrolysis of lactose takes
place. Furthermore, .beta.-D-galactosidase is the key enzyme in the
case of the breakdown of glycolipids, mucopolysaccharides and
glycoproteins. As further physiologically important glycosidases,
there may be mentioned .alpha.-D-galactosidase, .alpha.-D- and
.beta.-D-glucosidase and also .alpha.-D-mannosidase.
Over and above their physiological value, in recent years, the
glycosidases have achieved importance in the diagnostic as well as
in the biotechnological fields. Thus, for example, these enzymes
are used to an increasing extent as indicator enzymes for enzyme
immunoassays. In this connection, .beta.-D-galactosidase is
especially preferred.
The presence of the enzyme N-acetyl-.beta.-D-glucosaminidase
(.beta.-NAGase) in body fluids is a valuable indicator for diseases
or impaired functions in the organism. In the urine, for example,
increased values in the case of kidney transplants are an
indication of the rejection of the donor kidney. Increased values
also occur in the case of a number of diseases and of toxic damage
of the kidneys. In the saliva of females, the NAGase activity is an
indicator of fertility and pregnancy.
For the determination of the activity of hydrolases, the
enzyme-containing sample is reacted with an appropriate substrate.
The substrate is cleaved by the enzyme and one of the cleavage
products is detected in an appropriate manner. The natural
substrates of the enzyme to be detected are often suitable as
substrates. However, especially preferred are chromogenic compounds
in which one of the cleavage products is a residue which can be
detected in the spectroscopically visible or in the ultra-violet
region.
As chromogenic substrates for the determination enzymes cleaving
ester and glycosidic bonds, in published European Patent
Specification No. A-0,274,700 U.S. Pat. No. 4,900,822 there are
described dihydroresorufin compounds. After enzymatic cleavage of
the substrates, readily water-soluble leuko coloured materials
arise which can be oxidised easily by oxidation agents to give
coloured materials. Disubstituted dihydroresorufin derivatives are
hydrolysed in a multi-step reaction sequence which can give rise to
complications in the case of kinetic measurements.
A large number of chromogenic substrates are known for the
determination of glycoside bond-cleaving enzymes. Thus, in Biochem.
Z., 333, 209/1960, there are described phenyl-.beta.-D-galactoside,
as well as some further derivatives substituted on the aromatic
ring (for example o-nitrophenyl- and
p-nitrophenyl-.beta.-D-galactosides) as substrates for
.beta.-D-galactosidase. The phenols liberated by hydrolysis are
determined photometrically in the ultraviolet range or, in the case
of nitrophenols, in the shortwave visible wavelength range. An
oxidative coupling with aminoantipyrine can also follow as
indicator reaction (see Analytical Biochem., 40, 281/1971).
For histochemical investigations, naphthyl-.beta.-D-galactosides
are used, for example 1-naphthyl compounds in Histochemie, 35,
199/1973, the 6-bromo-2-naphthyl derivative in J. Biol. Chem., 195,
239/1952 and naphthol-.beta.-D-galactoside in Histochemie, 37,
89/1973. For visualisation, the naphthols thereby formed are
reacted with various diazonium salts to give azo coloured
materials. In the case of such necessary coupling reactions, sample
components can, for example, have a disturbing effect when they
react with the substances liberated by the enzymatic cleavage
instead of the coupling components.
For the detection of the enzyme N-acetyl-.beta.-D-glucosaminidase
(NAGase), it is suggested in U.S. Pat. No. 3,968,011 to use those
phenol derivatives which are cleaved at the pH values necessary for
the enzyme reaction and the reaction products of which form a
colour or can be detected or measured at a basic pH value.
Therefore, a rebuffering is necessary.
In the same way, in published European Patent Specification No.
0,097,506, NAGase substrates are suggested which, at an acidic pH
value, liberate, in the presence of the enzyme, a chromogen, for
example p-nitrophenol, which, after rebuffering to a basic pH
value, forms a colour which can be measured. In the case of the
liberation of umbelliferone, this can be detected fluorimetrically
which is, however, very expensive for apparatus and, especially in
the case of biological samples, is also subject to disturbances
because of the fluorescent background present therein.
In published European Patent Specification No. 0,060,793, U.S. Pat.
No. 4,433,139, sulphophthaleinyl-N-acetyl-.beta.-D-glucosaminides
are described as substrates for NAGase. However, the necessity of
the measurement of a blank value and the additional handling step
which is necessary in order to rebuffer the reaction mixture is
disadvantageous.
Published European Patent Specification No. 0,180,961, U.S. Pat.
No. 4,754,025, discloses, as .beta.-NAGase substrates,
N-acetyl-.beta.-D-glucosaminides substituted in the 2- and
4-positions by halogen or nitro. The process for the determination
of NAGase by means of these substrates can admittedly be carried
out without additional handling steps and without blank value
measurements but the solubility of the substrate is very low even
in the presence of wetting agents. In the case of the measurement
of NAGase in urine, this results in disturbances due to urine
components and, in the case of low enzyme concentrations, the time
required for carrying out the test is very great.
Sodium
3,3'-dichlorophenolsulphonphthaleinyl-N-acetyl-.beta.-D-glucosamidine
is described in published European Patent Specification No.
0,294,804. Here, too measurements can be carried out directly and
without stopping but the detection reaction must be carried out at
pH 6.25 since only from there but not at the acidic pH value is the
liberated chromogen sufficiently coloured. Since the enzyme in the
reaction rate at pH 4.5 to 5 shows a maximum, this results in a
retarded enzymatic cleavage of the substrate. In addition, for all
previously described analysis processes for the determination
NAGase, a spectrophotometric apparatus is necessary.
Substrates which also permit a visual assessment, either directly
or after alkali treatment, are described in Federal Republic of
Germany Patent Specification No. 28 57 145, U.S. Pat. No.
4,318,986. The coupled coloured materials have the general formula
HX-A-Y-NO.sub.2, wherein A is an aromatic nucleus, HX is an
auxochromic group and Y is an unsaturated radical. These substrates
liberate compounds which indicate the enzyme by a red or blue
coloration. However, the process has the disadvantage that, in the
presence of the enzyme, only a colour change takes place since the
substrates themselves are coloured. In the case of non-pretreated
or diluted samples, for example urine, the substrates must be
present in high concentrations since otherwise components of the
urine drastically slow down the enzymatic cleavage. Even substrates
with a weak inherent colour give strongly coloured test strips in
the case of the necessary concentrations. A detection via a colour
change makes low enzyme concentrations non-detectable or only
detectable after a very long period of time.
In published Japanese Patent Specification No. A-01 068389, N- and
O-substituted aminophenyl derivatives are suggested as NAGase
substrates which, as N-substituents, carry a phenol or naphthol
radical with free hydroxyl groups or a quinonimine structure. As a
condition, it is stated that one of the two N-substituents present
in the molecule can exclusively carry electron-attracting radicals,
whereas the other one is substituted by electron donors. These
compounds have a very low water solubility. Consequently, they are
very subject to disturbance due to sample components. For example,
the described compounds are not cleaved by HAGase in urine.
Summarising, the disadvantages of the hydrolase substrates known
from the prior art can be stated as follows: Substrates which
permit a multiple enzymatic cleavage complicate the kinetic
measurement. A light absorption of the radical split off by the
enzyme to be determined in the short-wave visible wavelength range
(for example yellow-coloured radicals) is, in many samples and
especially those of body fluids where many substances are present
which also absorb in the short-waved wavelength range, not to be
determined free from disturbances. The making visible of a radical
which can be split off enzymatically by coupling with a further
compound can also be disturbed by sample components. Fluorimetric
determinations are expensive for apparatus and, especially in the
case of biological samples, are often disturbed by a background
fluorescence of the sample material. With hydrolase substrates
which, in the case of enzymatic cleavage, merely change their
colour or pass through a colour change, determinations of enzymes
of low concentration are often only possible with high substrate
concentrations, for their reaction much time is required and, under
certain circumstances, they can inhibit the enzyme to be
determined. Sparing solubility of the hydrolase substrate often
leads to a very low sensitivity of the method of determination and
increases the time required until the desired result is obtained.
Determination processes with hydrolase substrates also require the
additional steps such as blank value measurement or rebuffering of
the reaction mixture, which requires relatively too much time and,
in addition, the carrying out thereof is laborious and, in some
cases, complicated so that only experts but not lay persons can
carry them out satisfactorily and reproducably. Furthermore,
determination methods which require additional handling steps
cannot be applied to or can only be applied with difficulty to test
carriers, i.e. so-called dry chemical tests, and be carried out
with such. Furthermore, determination processes which require, for
example, a rebuffering step, do not permit kinetic determinations
but rather are purely end-point methods.
The present invention is here helpful. The present invention, as it
is hereinafter described and claimed, solves the problem of making
available hydrolase substrates which make the determination of
hydrolases quick and simple, which permit not only kinetic
measurement but also end point determinations, which are also
capable of sensitively determining low enzyme concentrations, which
can serve for a hydrolase determination which is as free as
possible of disturbances, which do not make any expensive apparatus
necessary and which can also be used on test carriers.
Thus, the present invention is concerned with the use of N- and
O-substituted aminophenol derivatives of the general formula:
##STR2## wherein G is an organic or inorganic acid residue or a
glycoside radical, R.sup.1 and R.sup.2, which are the same or
different, are hydrogen or halogen atoms, SO.sub.3 H, PO.sub.3
H.sub.2 or a salt of these acid groups, hydroxyl, nitro, carboxyl,
carboxamido or cyano or an alkyl, alkenyl, alkoxy, alkylsulphinyl,
alkylsulphonyl, alkoxycarbonyl, alkylcarbonyl, aryl or aralkyl
radical optionally substituted one or more times by hydroxyl,
carboxyl, halogen, cyano, SO.sub.3 H or PO.sub.3 H.sub.2 residues
or a salt of these acid residues or, when two substituents are
present on neighbouring carbon atoms, these can together present a
1,4-butadiendiyl radical which is optionally substituted one or
more times by SO.sub.3 H, PO.sub.3 H.sub.2 or a salt of these acid
residues, on alkyl and/or a carboxyl group, R.sup.3 is a hydrogen
atom, CO--COOH, SO.sub.3 H, PO.sub.3 H.sub.2 or a salt of these
acid residues, an alkylcarbonyl radical optionally substituted one
or more times by halogen, COOH, SO.sub.3 H and/or PO.sub.3 H.sub.2
or a salt of these acid groups or an arylcarbonyl radical
optionally substituted one or more times by SO.sub.3 H, PO.sub.3
H.sub.2 or a salt of these acid residues and L is a radical of the
general formula: ##STR3## wherein R.sup.4 and R.sup.5, which can be
the same or different, are alkyl radicals or together represent a
saturated hydrocarbon chain containing 3 to 6 members which can be
interrupted by oxygen, sulphur or nitrogen, whereby alkyl or the
hydrocarbon chain is optionally substituted one or more times by
hydroxyl, carboxyl, alkoxycarbonyl, alkoxy, SO.sub.3 H or PO.sub.3
H.sub.2 groups, a salt of these acid residues or halogen and
R.sup.6 and R.sup.7, which can be the same or different, are
hydrogen or halogen atoms, hydroxyl, carboxamido or an alkyl,
alkoxy, alkylcarbonyl, alkoxycarbonyl, aryl or aralkyl radical
optionally substituted one or more times by hydroxyl, carboxyl,
halogen, SO.sub.3 H or PO.sub.3 H.sub.2 or a salt of these acid
residues or L is a pyrazolo-heterocyclic radical of the general
formula: ##STR4## in which X-Y is NR.sup.8 --CO or N.dbd.CR.sup.9,
in which R.sup.8 is an hydrogen atom or an alkyl radical and
R.sup.9 is alkyl, alkenyl, alkoxy, alkylthio, aryl, aralkyl, in
each case optionally substituted by hydroxyl, dialkylphosphinyl,
carboxyl, SO.sub.3 H, PO.sub.3 H.sub.2, a salt of these acid
residues and/or alkoxycarbonyl; amino, which is optionally
substituted by one or two alkyl radicals which, in turn, are
optionally substituted one or more times by hydroxyl, carboxyl
and/or alkoxycarbonyl, and when amino is substituted by two alkyl
radicals, these can be joined to form a ring which, apart from the
nitrogen atom of the amino group, can optionally also be
interrupted by oxygen, sulphur or a further nitrogen atom or amino
can optionally be substituted by one or two acyl radicals, alkoxy-
and/or aralkoxycarbonyl radicals, H.sub.2 N--CO, alkyl-, aralkyl-
and/or arylcarbamoyl radicals; or is hydrogen, carboxyl,
alkoxycarbonyl, carboxamido or halogen and Z is NR.sup.10
--N.dbd.N, in which R.sup.10 is a hydrogen atom or an alkyl or
aralkyl radical or Z is an unsaturated chain containing 3 to 5
members of nitrogen atoms or of carbon atoms and optionally one or
more nitrogen or sulphur atoms, whereby carbon atoms are optionally
substituted by alkyl, alkoxy, hydroxyalkyl, alkylthio, hydroxyl,
aralkyl, aryl, carboxyl, carboxamido, alkoxycarbonyl, cyano, amino,
which is optionally substituted by one or two alkyl radicals which,
in turn, are optionally substituted by one or more hydroxyl,
carboxyl and/or alkoxycarbonyl radicals, and/or halogen, as well as
nitrogen atoms which are not connected via a double bond are
optionally substituted by alkyl or aralkyl or two neighbouring
chain substituents optionally form an alkylene radical which, in
turn, is optionally substituted or anellated with aryl, and the
corresponding tautomeric radicals thereof, as hydrolase
substrates.
From published British Patent Specification No. 2,061,537 and from
published Federal Republic of Germany Patent Specification No. 22
60 202 U.S. Pat. No. 3,952,009 are known pyrazolotriazole
derivatives as coloured material formers for the production of
photographic pictures which correspond to general formula (I) when
there G is an alkanecarboxylic acid radical, R.sup.3 is a hydrogen
atom, an arylcarbonyl radical or an alkylcarbonyl radical
optionally substituted one or more times by halogen and L is a
pyrazoloheterocyclic radical of general formula (III) in which X-Y
is n=CR.sup.9, R.sup.9 has the above-given meaning, and in addition
Z is a 1,2,4-triazole ring in which a nitrogen atom not connected
via a double bond is substituted by a hydrogen atom. The other
compounds of general formula (I) are new.
Thus, the present invention also provides N- and C-substituted
aminophenol derivatives of the general formula: ##STR5## in which G
is the residue of an organic or inorganic residue or a glycoside
radical, R.sup.1 and R.sup.2, which can be the same or different,
are hydrogen, halogen, SO.sub.3 H, PO.sub.3 H.sub.2 or a salt of
these acid groups, a hydroxyl, nitro, carboxyl, carboxamido or
cyano group or an alkyl, alkenyl, alkoxy, alkylsulphinyl,
alkylsulphonyl, alkoxycarbonyl, alkylcarbonyl, aryl or aralkyl
radical optionally substituted one or more times by hydroxyl,
carboxyl, halogen, cyano, SO.sub.3 H or PO.sub.3 H.sub.2 or a salt
of one of these acid residues or, when both substituents are
present on neighbouring carbon atoms, together represent a
1,4-butadiendiyl radical which is optionally substituted one or
more times by SO.sub.3 H, PO.sub.3 H.sub.2 or a salt of these acid
residues, R.sup.3 is hydrogen, CO--COOH, SO.sub.3 H, PO.sub.3
H.sub.2 or a salt of these acid residues, an alkylcarbonyl radical
optionally substituted one or more times by halogen, COOH, SO.sub.3
H and/or PO.sub.3 H.sub.2 or a salt of these acid residues or an
arylcarbonyl radical optionally substituted one or more times by
SO.sub.3 H, PO.sub.3 H.sub.2 or a salt of these acid residues and L
is a radical of the general formula: ##STR6## in which R.sup.4 and
R.sup.5, which can be the same or different, are alkyl radicals or
together represent a saturated hydrocarbon chain containing 3 to 6
members which can be interrupted by oxygen, sulphur or nitrogen,
whereby alkyl or the hydrocarbon chain is optionally substituted
one or more times by hydroxyl, carboxyl, alkoxycarbonyl, alkoxy,
SO.sub.3 H or PO.sub.3 H.sub.2 groups, a salt of these acid
residues or halogen and R.sup.6 and R.sup.7, which can be the same
or different, are hydrogen halogen, hydroxyl or carboxamido or an
alkyl, alkoxy, alkylcarbonyl, alkoxycarbonyl, aryl or aralkyl
radical optionally substituted one or more times by hydroxyl,
carboxyl, halogen, SO.sub.3 H or PO.sub.3 H.sub.2 or a salt of one
of these acid residues or L is a pyrazolo-heterocyclic radical of
the general formula: ##STR7## in which X-Y is NR.sup.8 --CO or
N.dbd.CR.sup.9, whereby R.sup.8 is a hydrogen atom or an alkyl
radical and R.sup.9 is alkyl, alkenyl, alkoxy, alkylthio, aryl
aralkyl, optionally in each case substituted by hydroxyl,
dialkylphosphinyl, carboxyl, SO.sub.3 H, PO.sub.3 H.sub.2, a salt
of one of acid residues and/or alkoxycarbonyl; amino, which is
optionally substituted by one or two alkyl radicals which, in turn,
are optionally substituted one or more times by hydroxyl, carboxyl
and/or alkoxycarbonyl, whereby, when amino is substituted by two
alkyl radicals, these residues can also be joined to form a ring
which, apart from the nitrogen atom of the amino group, can
optionally also be interrupted by oxygen, sulphur or a further
nitrogen atom or amino is optionally substituted by one or two acyl
radicals, alkoxy- and/or aralkoxycarbonyl radicals, H.sub.2 N--CO,
alkyl-, aralkyl- and/or arylcarbamoyl radicals; or is hydrogen,
carboxyl, alkoxycarbonyl, carboxamido or halogen, and Z is
NR.sup.10 --N.dbd.N, whereby R.sup.10 is hydrogen, alkyl or aralkyl
or Z is an unsaturated chain containing 3 to 5 members of nitrogen
atoms or of carbon atoms and optionally one or more nitrogen or
sulphur atoms, whereby carbon atoms are optionally substituted by
alkyl, alkoxy, hydroxyalkyl, alkylthio, hydroxyl, aralkyl, aryl,
carboxyl, carboxamido, alkoxycarbonyl, cyano, amino, which is
optionally substituted by one or two alkyl radicals which, in turn,
are optionally substituted one or more times by hydroxyl, carboxyl
and/or alkoxycarbonyl, and/or halogen, as well as nitrogen atoms
which are not connected via a double bond are optionally
substituted by alkyl or aralkyl or two neighbouring chain
substituents optionally form an alkylene radical which, in turn, is
optionally substituted or anellated with aryl, and the
corresponding tautomeric radicals, with the proviso that when G is
an alkanecarboxylic acid radical, R.sup.3 is a hydrogen atom, an
arylcarbonyl radical or an alkylcarbonyl radical optionally
substituted one or more times by halogen and L is a
pyrazolo-heterocyclic radical of general formula (III) in which X-Y
is N.dbd.CR.sup.9, whereby R.sup.9 has the same meanings as above,
Z does not form a 1,2,4-triazole ring in which a nitrogen atom not
connected via a double bond is substituted with hydrogen.
The present invention also provides a process for the preparation
of these compounds, wherein a compound of the general formula:
in which G is the residue of an organic or inorganic acid or a
glycoside radical and D is a reactive group, whereby functional
groups in G are optionally protected with protective groups such as
are conventional in peptide and carbohydrate chemistry, is reacted
with a compound of the general formula: ##STR8## in which R.sup.1
-R.sup.3 and L have the meanings given in general formula (I) and
R.sup.3 can additionally be an amino protective group and A is a
hydrogen atom, an optionally substituted ammonium ion or an alkali
metal, and protective groups are optionally subsequently split
off.
Furthermore, the present invention provides compounds of the
general formula V' ##STR9## wherein R.sup.1 -R.sup.3 have the
meanings given in general formula I and L is a
pyrazolo-heterocyclic radical of the general formula: ##STR10## in
which X-Y and Z have the meanings given in general formula (I) with
the proviso that when R.sup.3 is an arylcarbonyl radical or an
alkylcarbonyl radical optionally substituted one or more times by
halogen and X-Y is C.dbd.NR.sup.9, Z does not form a 1,2,4-triazole
ring in which a nitrogen atom not connected via a double bond is
substituted by hydrogen.
In addition, the present invention provides a process for the
preparation of compounds of general formula (V'), wherein
a) a compound of the general formula: ##STR11## in which R.sup.1,
R.sup.2 and L have the meanings given in general formula (V'), is
reduced and, when R.sup.3 is not to be a hydrogen atom, the anilino
group is acylated and an ester group optionally present is split
off; or
b) a compound of general formula (I), in which L is a
pyrazolo-heterocyclic radical of the general formula: ##STR12## in
which X-Y, G and Z have the meanings given in general formula (I),
is reacted with an appropriate hydrolase.
The present invention also provides compounds of the general
formula: ##STR13## in which R.sup.1 and R.sup.2 have the meanings
given in general formula (I) and L is a pyrazolo-heterocyclic
radical of the general formula: ##STR14## in which X-Y and Z have
the meanings given in general formula (I).
Furthermore, the present invention provides a process for the
preparation of compounds of general formula (VII), wherein
a) a compound of general formula (I), in which L is a
pyrazol-heterocyclic radical of the general formula: ##STR15## in
which X-Y and Z have the meanings given in general formula (I), is
reacted with a hydrolase and the reaction product is oxidised
or
b) a compound of the general formula:
in which L is a pyrazolo-heterocyclic radical of the general
formula: ##STR16## in which X-Y and Z have the meanings given in
general formula (I) is reacted with a phenol of the general
formula: ##STR17## in which R.sup.1 and R.sup.2 have the meanings
given in general formula (VII) and V is a hydrogen or halogen atom
or a COOH or SO.sub.3 H group, in the presence of an oxidation
agent, or
c) a compound of the general formula:
in which L is a pyrazolo-heterocyclic radical of the general
formula: ##STR18## in which X-Y and Z have the meanings given in
general formula (I), is reacted with a compound of the general
formula: ##STR19## in which R.sup.1 and R.sup.2 have the meanings
given in general formula (VII) and Hal is a halogen atom, or
d) a compound of the general formula:
in which E is a nitro or nitroso group and L is a
pyrazolo-heterocyclic radical of the general formula: ##STR20## in
which X-Y and Z have the meanings given in general formula (I), is
reacted with an organo-metallic compound of the general formula:
##STR21## in which R.sup.1 and R.sup.2 have the meanings given in
general formula (VII), Q is a hydroxyl group or a dialkylamino
radical and Me is lithium or magnesium substituted by a halogen
atom, and subsequently worked up in an aqueous medium.
The subject of the present invention is also the use of compounds
of general formulae (V') and (VII) for the preparation of new
compounds of general formula (I).
The subject of the present invention is also a process for the
colorimetric determination of a hydrolase by reaction of a
chromogenic enzyme substrate with the enzyme, oxidation of a leuko
coloured material liberated from the substrate by the enzyme and
determination of the resultant coloured material as a measure of
the amount of enzyme, wherein, as enzyme substrate, there is used a
compound of general formula (I).
In addition, a subject of the present invention is a diagnostic
agent for the determination of a hydrolase, containing a
chromogenic enzyme substrate and an appropriate buffer substance,
wherein, as chromogenic enzyme substrate, there is used a compound
of general formula (I).
Finally, a subject of the present invention is the use of a
compound of general formula (I) for the production of a diagnostic
agent for the determination of a hydrolase.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a calibration curve at .lambda. max 560 nm for the
substrate from example 4e;
FIG. 2 shows a calibration curve at .lambda. max 573 nm for the
substrate of example 4g;
FIG. 3 shows the calibration curve at .lambda. max 525 nm for the
substrate of example 4(h);
FIG. 4 shows the calibration curve at .lambda. max 565 nm for the
substrate of example 5;
FIG. 5 shows the test carrier having paper, mesh, covering mesh and
plastic film; and
FIG. 6 is a calibration curve having known enzyme-containing
solutions.
FIG. 7 is a calibration curve having known
.beta.-galactosidase-containing solutions.
We have found that compounds of general formula (I) with the
above-given meaning are hydrolase substrates which solve the
defined problems in an outstandingly good manner.
By an inorganic acid residue in the definition of G, there is to be
understood, in particular, the ortho- and pyrophosphoric acid and
sulphuric acid residue which is attached to the aminophenol base
structure via an ester bond. Preferred are the residues PO.sub.3
MM' and SO.sub.3 M and especially PO.sub.3 MM', whereby, in the
case of the free acids, M and M' are hydrogen atoms, whereas, when
the acids are present as salts, M and M' stand for alkali metal,
alkaline earth metal or ammonium ions.
By alkali metal ions in the definitions of M and M', there are to
be understood, in particular, lithium, sodium and potassium ions.
Alkaline earth metal ions are, in particular, magnesium, calcium or
barium ions.
Ammonium ions in the definitions of M and M' can be the
unsubstituted ammonium ion NH.sub.4.sup.+ or ammonium ions
substituted one or more times by alkyl or aralkyl radicals. The
alkyl radical is hereby to be understood to be one containing up to
6 carbon atoms, the methyl or ethyl radical being preferred. By an
aralkyl radical is to be understood one in which the above-defined
alkyl radical is substituted by an aryl radical, whereby aryl is a
carbon aromatic or hetero-aromatic radical and preferably one
containing 6 to 10 ring atoms, especially a phenyl or naphthyl
radical. The preferred aralkyl radical is a benzyl radical. The
substituents of substituted ammonium ions can be the same or
different. As ammonium ions there can also be used cations of
quaternised nitrogen heterocyclic compounds, examples therefor
including a piperidinium cation and the pyridinium ion.
By an organic acid residue in the definition of G are to be
understood, in particular, the residues of alkanecarboxylic acids,
amino acids and oligopeptides which, with the carboxyl end thereof,
are present bound to the aminophenol base structure of general
formula (I) as esters.
Alkanecarboxylic acid residues in the definition of G are compounds
containing up to 20 carbon atoms. Especially preferred are acetic
acid, propionic acid, butyric acid, palmitic acid and stearic acid.
Besides saturated acid residues, G can also be an unsaturated acid
residue, for example of oleic acid, linoleic acid or linolenic
acid.
An amino acid residue is preferably the residue of a
naturally-occurring .alpha.-amino acid in its L- or D-form or also
in the racemic form thereof. Especially preferred are the residues
of glycine, alanine, valine, leucine, isoleucine, phenylalanine and
tyrosine, in which, in each case, the L-form is quite especially
preferred.
By an oligopeptide residue is to be understood, for example, a di-,
tri-, tetra- or pentapeptide and preferably a di- or tripeptide,
whereby the amino acid components are preferably the
above-mentioned amino acids.
The amino groups of the amino acids and oligopeptide residues,
bound in the manner of an ester to the aminophenol radical, can be
present in free or protected form. As protective groups, there are
here to be understood all the conventional amino protective groups,
especially acyl, oxycarbonyl, thiocarbonyl, sulpho, sulphino,
vinyl, cyclohexenyl, phosphoryl and carbamoyl groups. Especially
preferred amino protective groups include the tosyl,
benzyloxycarbonyl and tert.-butoxycarbonyl radicals.
A glycoside radical in the definition of G can be a mono- or
oligosaccharide. The sugar residue can be bound to the aminophenol
base structure .alpha.- or .beta.-glycosidically. Examples of
preferred monosaccharides include galactose, glucose and mannose.
N-acetylglucosamine is especially preferred. Quite especially
preferred is .beta.-glycosidically bound
N-acetyl-2-D-glucosamine.
However, oligosaccharides have also proved to be suitable as sugar
residues. As oligosaccharides are especially designated those which
are made up to 2 to 10 and preferably 2 to 7 monosaccharide units.
The heptaoses are especially preferred.
From the group of the organic and inorganic acid residues and of
the glycoside residues in the meaning of G, the glycoside residues
are preferred for compounds of general formula I. The
N-acetylglucosamine residue is especially advantageous.
Insofar as nothing is stated to the contrary, the following
radicals have the following means in the general formulae used
herein: "alkyl", also in alkylsulphinyl, alkylsulphonyl,
alkylcarbonyl, alkylthio, alkylcarbamoyl, alkylamino and aralkyl
radicals, means a straight-chained or branched alkyl radical
containing up to 6 and preferably up to 4 carbon atoms, examples
therefor including the methyl, ethyl, propyl, isobutyl and
tert.-butyl radicals.
When an amino group is substituted by two alkyl radicals, these
radicals can be joined to give a ring which, in all, represents a
ring interrupted by a nitrogen atom. Preferred are hereby those
amino groups which, in all, represent a five- or six-membered ring
and which, in turn, is optionally interrupted by oxygen, sulphur or
a further nitrogen atom. The morpholino radical is especially
preferred.
A hydroxyalkyl radical is an alkyl radical containing up to 6 and
preferably up to 4 carbon atoms substituted by a hydroxyl group.
The hydroxyalkyl radical can be the residue of a primary, secondary
or tertiary alcohol. Especially preferred are the 2- and
1-hydroxyethyl and the hydroxymethyl radicals.
"Alkoxy", also in alkoxy- and arylkoxycarbon radicals, stands for a
straight-chained or branched alkoxy radical containing up to 6 and
preferably up to 4 carbon atoms. Examples therefor include the
methoxy, ethoxy, propoxy, isobutoxy and tert.-butoxy radicals.
"Aryl", also in arylcarbonyl and arylcarbamoyl radicals, indicates
a carbon aromatic or heteroaromatic radical and preferably one with
6 to 10 ring atoms, especially a phenyl or naphthyl radical, which,
in addition, can also be substituted by alkyl, alkoxy and/or
halogen, the phenyl radical being especially preferred.
An "aralkyl" radical, also in an aralkylcarbamoyl radical, means a
radical in which an alkyl radical as defined hereinbefore is
substituted by an aryl radical, the benzyl radical being
preferred.
An "aralkoxy" radical, for example an aralkoxycarbonyl radical,
signifies a radical in which an alkoxy radical as hereinbefore
defined is substituted by an aryl radical, the benzyloxy radical
being preferred.
"Halogen" stands for a fluorine, chlorine, bromine or iodine atom,
fluorine and chloride being preferred.
"Alkenyl" means an unsaturated hydrocarbon radical containing 2 to
6 and preferably 2 to 4 carbon atoms, examples thereof including
the vinyl and allyl radicals.
An acyl radical designates a carboxylic acid residue which can
contain alkyl, aralkyl or aryl radicals, the acetyl, phenylacetyl
and benzoyl radicals being preferred. By an alkylene is to be
understood a straight-chained or branched, saturated or unsaturated
hydrocarbon radical containing 3 to 5 and preferably 3 or 4 carbon
atoms with two free bonding positions. Examples thereof include
##STR22## --CH.dbd.CH--CH.dbd.CH--, the butadiendiyl radical
(--CH.dbd.CH--CH.dbd.CH) and the tetramethylene radical
(--(CH.sub.2).sub.4 --) being preferred.
By an dialkylphosphinyl group is to be understood the residue
##STR23## whereby alkyl has the same meanings given above. The
dimethylphosphinyl residue is preferred.
As salts of SO.sub.3 H, PO.sub.3 H.sub.2 and carboxyl residues,
there can be used alkali metal, alkaline earth metal or ammonium
salts. By alkali metal salts are to be understood lithium, sodium,
potassium, rubidium and caesium salts, whereby lithium, sodium and
potassium salts and especially sodium and potassium salts are
preferred. Alkaline earth metal salts are those of beryllium,
magnesium, calcium, strontium and barium, whereby magnesium and
calcium salts and especially calcium salts are preferred. As
ammonium salts, there can be used those of the unsubstituted
ammonium ion NH.sub.4 +. However, it is also possible to use those
ammonium salts in which the ammonium ion is substituted by up to 4
alkyl, aryl or aralkyl radicals. For these radicals there apply the
above-given definitions, whereby the alkyl radical is preferably
the methyl, ethyl or n-propyl radical, the aryl radical is
preferably the phenyl radical and the aralkyl radical is preferably
the benzyl radical. As ammonium ions there can also be used cations
of quaternised nitrogen heterocyclic compounds, examples thereof
including the piperidinium cation and the pyridinium ion.
As carboxamido radical, there is to be understood the CONH.sub.2
group but also those groups in which the amino group is also
substituted by one or two alkyl radicals which optionally are, in
turn, substituted by one or more hydroxyl, carboxyl and/or
alkoxycarbonyl radicals.
Advantageous are, in particular, those compounds according to the
present invention in which R.sup.3 is a hydrogen atom or an
alkylcarbonyl radical substituted one or more times by halogen, for
example trifluoroacetyl. The meaning of hydrogen for R.sup.3 is
quite especially preferred.
Compounds according to the present invention in which L is a
pyrazolo-heterocyclic radical of general formula (III) are
preferred. Of these, especially those in which Z is so positioned
that at least one double bond of the unsaturated chain is in
conjugation with the double bond or with the nitrogen atom in
general formula (III).
Furthermore, compounds according to the present invention in which
L is a pyrazolo-heterocyclic radical of general formula (III) are
especially preferred which, in the unsaturated chain Z, if this
contains nitrogen atoms which are not connected via a double bond,
are only substituted with alkyl or aralkyl radicals on these
nitrogen atoms.
Tautomeric forms are also possible for radicals of general formula
(III). These are also to be regarded as being covered by general
formula (III).
According to the present invention, those compounds of general
formula (I) are preferred in which L is a radical of one of the
following general formulae (XIII) to (XXIV): ##STR24## as well as
possibly the corresponding tautomeric forms. In the above general
formulae, X-Y and R.sup.10 have the same meanings as hereinbefore.
R.sup.11, R.sup.12, R.sup.13 and R.sup.14, which can be the same or
different, stand for hydrogen, hydroxyl, alkyl, alkoxy, alkylthio,
aralkyl, aryl, carboxyl, alkoxycarbonyl, carboxamido, cyano, amino,
which is optionally substituted once or twice by alkyl radicals
which, in turn, are optionally substituted by one or more hydroxyl,
carboxyl and/or alkoxycarbonyl groups, or halogen, whereby two
neighbouring radicals optionally form an alkylene radical which, in
turn, is optionally substituted or anellated with aryl. The
definitions of the radicals correspond to those given
hereinbefore.
According to the present invention, compounds of general formula
(I) are especially preferred in which L is a radical of the groups
of general formulae (XIII), (XIV), (XV), (XVII), (XVIII) and (XX)
and optionally also the corresponding tautomeric forms thereof.
Quite especially preferred are those compounds in which X-Y is
N.dbd.CR.sup.9, wherein R.sup.9 can have the meaning given in
general formula (III) but is preferably a hydrogen atom or an
alkoxy radical.
Outstandingly preferred in the meaning of the present invention is
especially the .beta.-glycosidically-bound
N-acetyl-2-D-glucosaminide of
4-hydroxyphenyl-2-methylpyrazolo-[1,5-a]pyridin-3-ylamine.
The compounds of general formula (I) are new. They can be prepared
by reacting a leuko methine coloured material of the general
formula: ##STR25## in which R.sup.1 -R.sup.3 and L have the same
meanings as given in general formula (I) and A is a hydrogen atom,
an optionally substituted ammonium ion or an alkali metal, with a
compound of the general formula:
in which G is the residue of an organic or inorganic acid or a
glycoside residue having the meanings given in general formula (I),
whereby functional groups present in the glycoside residue, for
example amino and/or hydroxyl groups, are optionally substituted
with protective groups which are conventional in peptide and
carbohydrate chemistry, and D is a reactive group, whereafter
protective groups are optionally split off.
An unsubstituted ammonium ion is to be understood to be
NH.sub.4.sup.+. This ion can optionally be substituted one or more
times by alkyl or aralkyl radicals with meanings given for A in
general formula (V). The substituents of substituted ammonium ions
can be the same or different. As ammonium ions, there can also be
used quaternised nitrogen-heterocyclic compounds, examples therefor
including the piperidinium cation and the pyridinium ion.
As alkali metals in the meaning of A in general formula (V), there
can be used lithium, sodium and potassium, sodium being
preferred.
D means a reactive group which is able to react with the phenol or
phenolate group OA of general formula (V). The choice of the
reactive group depends upon the nature of the radical G. If G is a
sugar residue, then D is preferably a group which can readily be
replaced, for example an acetyl radical or a halogen atom, which
can be selected from fluorine, chlorine, bromine and iodine,
chlorine, bromine and iodine being preferred.
As protective groups which are conventional in carbohydrate
chemistry, there are to be mentioned, in particular, the acetyl,
benzoyl, benzyl and trimethylsilyl radicals.
When G is a residue of an amino acid or of a peptide which is to
esterified with its carboxyl end with an aminophenol of general
formula (V), then, as reactive group D, there can be used all
groups which are conventional in peptide chemistry. As reactive
derivatives, there can be used, for example, the acid halides,
preferably the acid chloride, or the mixed anhydrides or active
esters usually employed in peptide syntheses. The same reactive
groups can also be used for the binding of alkanecarboxylic acids
to the aminophenol structure.
When G is an inorganic acid residue, compounds of general formula
(V) are preferably reacted with the corresponding acid halides and
especially acid chlorides.
In every case, in the case of the esterification, care is to be
taken, when R.sup.3 in general formula (V) is a hydrogen atom, to
substitute this amino group, before carrying out the esterification
reaction, with a protective group, for example a group
conventionally used for this purpose in peptide chemistry,
whereafter the protective group is again removed.
By way of example, the process for the preparation of compounds of
general formula (I) is to be illustrated using the example of the
especially preferred compounds in which G is an
N-acetyl-.beta.-D-glucosaminide radical. This process can also be
used correspondingly for the preparation of other glycoside
derivatives of general formula (I).
N-Acetyl-.beta.-D-glucosaminidyl derivatives of general formula (I)
according to the present invention can be prepared by reacting a
compound of the general formula: ##STR26## wherein W is a halogen
atom, R is a hydroxy protective group conventional in carbohydrate
chemistry, B is an azide group, a protected amino group or
NH--COCH.sub.3 or B and W together signify the group: ##STR27##
with a compound of the general formula: ##STR28## wherein R.sup.1
-R.sup.3 and L have the meanings given for general formula (I) and
A is a hydrogen atom, an optionally substituted ammonium ion or an
alkali metal, when B is a protected amino group, the amino
protective group is removed or when B is an azide group this is
converted by reduction into an amino group and the amino group is
converted by acetylation into an NHCOCH.sub.3 radical and finally
the hydroxy protective group is split off.
One possibility is, for example, to react a leuko methine coloured
material of the general formula: ##STR29## wherein R.sup.1 -R.sup.3
and L have the meanings given for general formula (I), with a
per-O-substituted L-halo-N-acetylglucosamine of the general
formula: ##STR30## wherein W is a halogen atom, B is NHCOCH.sub.3
and R is a protective group which is conventional in carbohydrate
chemistry, with Walden inversion on the C-1 atom of the sugar
residue, to give a per-O-substituted .beta.-glycoside, whereafter
the hydroxy protective groups are split off from the latter
according to known methods.
The reaction of compounds of general formulae (V) and (VI) with the
above-given meanings to give N-acetyl-.beta.-D-glucosaminides of
general formula (I) is preferably carried out in the presence of an
acid acceptor, for example an alkali metal hydroxide, carbonate or
bicarbonate, in aqueous acetone or under phase transfer conditions
in a water/benzene or water/chloroform mixture (cf. Synthesis,
223/1988).
Furthermore, the N-acetyl-.beta.-D-glucosaminides of general
formula (I) can be prepared by first converting the leuko coloured
materials of general formula (V), in which A is a hydrogen atom,
with an alkali metal hydroxide or alcoholate into the corresponding
alkali metal salt or with an optionally substituted amine into an
ammonium salt, whereby the alkali metal and the ammonium ion can
have the above-given meanings, whereafter these are then reacted in
a dipolar aprotic solvent, for example acetone, dimethyl
sulphoxide, dichloromethane of dimethylformamide, with the
per-O-substituted 1-halo-N-acetylglucosamines.
In addition, in the case of the synthesis of N-acetylglucosaminides
of general formula (I) from leuko coloured materials of general
formula (V) and 1-halo-N-acetylglucosamines, it has proved to be
useful to add individual silver salts or mixtures of silver salts
(silver oxide, carbonate on Celite.RTM., triflate, salicylate)
and/or individual mercury salts or mixtures of mercury salts
(mercury bromide, cyanide, acetate, oxide), optionally with the use
of a drying agent, for example calcium chloride or Drierit.RTM., in
a solvent, for example methylene chloride, chloroform, benzene,
toluene or dioxan.
There can also be used an oxazoline of general formula (VI),
wherein B and W together signify a group of the formula: ##STR31##
in the presence of an organic acid, for example p-toluenesulphonic
acid, or of a Lewis acid, for example boron trifluoride etherate or
ferric chloride, for the synthesis of N-acetylglucosaminides of
general formula (I). Examples of such glycosidation reactions are
described, for example, in Carbohydrate Research, 136, 309-323/1985
and 64, 334-338/1978.
Finally, processes for the preparation of N-acetylglucosaminides of
general formula (I) can be carried out in which, in the compound of
general formula (I), B is an amino group substituted with a
protective group, for example a benzyloxycarbonyl,
allyloxycarbonyl, dichloroacetamido or phthalimido radical, or a
substituent which is stable under the glycosidation conditions from
which an amino group can be liberated, for example an azide group.
The glycosidation reaction is carried out by splitting off the
protective groups according to methods of peptide chemistry or by
reduction of the azido group to free the amino group which is then
selectively N-acetylated in a final step (see, for example, J. Crg.
Chem., 32, 3767/1967).
The splitting off of protective groups takes place is the manner
known from carbohydrate chemistry by hydrogenolysis in the case of
protective groups of the benzyl type, by the action of sodium
methylate, sodium cyanide or sodium bicarbonate in methanol for the
splitting off of acyl radicals, for example acetyl radicals. The
methods for the splitting off of protective groups are described in
Adv. Carbohydr. Chem. Biochem., 39, 13/1981.
The synthesis of 1-halo-N-acetylglucosamines is described, for
example, in Org. Synth., Vol. 46, p. 1, Methods in Carbohydrate
Chem., 6, 282/1972 and in J. Org. Chem., 26, 445/1961.
The leuko coloured materials required for the preparation of
compounds of general formula (I) and which have the general
formula: ##STR32## wherein R.sup.1 -R.sup.3 and L have the meanings
given for general formula (I), whereby L is preferably a
pyrazolo-heterocyclic radical of the general formula: ##STR33## in
which X-Y and Z have the meanings given hereinbefore for the
corresponding compounds of general formula (I), are, with the
exception of those compounds according to published British Patent
Specification No. A-2,061,537 in which R.sup.3 is an arylcarbonyl
radical or an alkylcarbonyl radical optionally substituted one or
more times by halogen and in which Z is a 1,2,4-triazole ring, in
which a nitrogen atom not connected via a double bond is
substituted with hydrogen, are also new.
They can be prepared by reduction of the corresponding coloured
materials of the general formula: ##STR34## wherein R.sup.1,
R.sup.2 and L have the meanings given for compounds of general
formula (V'), according to known methods with reducing agents, for
example catalytic hydrogenation, sodium borohydride,
palladium/hydrazine or sodium dithionite. Such reducing agents are
described in Houben-Weyl, Vol. 4/lc and 4/ld.
Acyl radicals R.sup.3 such as are given for compounds of general
formula (I) can be introduced either at the stage of the leuko
coloured materials of general formula (V'), in which R.sup.3 is a
hydrogen atom, or at the stage of the protected per-O-substituted
N-acetylglucosaminide, as occurs in the case of the preparation of
compounds of general formula (I), in which G is a glycoside
residue. Activated acid derivatives, for example halides,
anhydrides and mixed anhydrides are used, such as are known from
peptide chemistry.
Starting from compounds of general formula (I), compounds of
general formula (V') can, of course, also be obtained by reaction
with an appropriate hydrolase.
The coloured materials of general formula (VII) necessary for the
preparation of compounds of general formula (V') are also new. They
are preferably obtained by the oxidative coupling of an amino
compound of the general formula:
in which L has the meaning given in general formula (I) but is
preferably a pyrazolo-heterocyclic radical of general formula (III)
in which X-Y and Z have the meanings given for compounds of general
formula (I), with a phenol of the general formula: ##STR35## in
which R.sup.1 and R.sup.2 have the meanings given in compounds of
general formula (I) and V is a hydrogen or halogen atom or a
carboxyl or SO.sub.3 H group.
For this purpose, an amino compound of general formula (VIII) and a
phenol of general formula (IX), preferably in which V is a hydrogen
atom, are reacted in the presence of an oxidation agent, for
example potassium ferricyanide, potassium peroxodisulphate,
potassium peroxomonosulphate, iodine, hydrogen peroxide/peroxidase,
lead dioxide, sodium hypochlorite, sodium hypobromite or an organic
oxidation agent, for example N-bromosuccinimide or a related
compound.
Furthermore, the coloured materials of general formula (VII) can
also be prepared by the reaction of N.sub.-- haloimines of the
general formula: ##STR36## in which R.sup.1 and R.sup.2 have the
meanings given for compounds of general formula (I) and Hal is a
halogen atom, whereby halogen is fluorine, chlorine, bromine or
iodine and preferably chlorine, with compounds of the general
formula:
in which L has the meaning given for general formula (I) but is
preferably a pyrazole-heterocyclic radical of general formula
(III), whereby X-Y and Z have the meanings given for general
formula (I).
The reaction conditions can be chosen analogously to those
described in Houben-Weyl, Vol. 7/3, pp. 296 et seq. Starting from
compounds of general formula (I), coloured materials of general
formula (VII) can also be obtained by reaction with an appropriate
hydrolase and subsequent oxidation. For the oxidation, there can,
in principle, be used the same substances which were described for
the oxidative coupling between compounds of general formula (VIII)
and those of general formula (IX).
Coloured materials of general formula (VII) can advantageously also
be obtained by reaction of a compound of the general formula:
in which E is a nitroso or nitro group and L is a
pyrazoloheterocyclic radical of general formula (III), whereby X-Y
and Z have the meanings given for general formula (I), with an
organo-metallic compound of the general formula: ##STR37## in which
R.sup.1 and R.sup.2 have the meanings given for general formula
(VII), Me is lithium or magnesium substituted by halogen and Q is a
hydroxyl group or a dialkylamino radical, and subsequent aqueous
working up of the reaction mixture. Radicals present possibly in
the compounds of general formulae (XXV) and (XXVI) which are not to
react with the Grignard or lithium compound are to be appropriately
protected. Information regarding such protective groups is to be
found, for example, in T. Greene, "Protecting Groups in Organic
Synthesis", pub. John Wiley and Sons, New York, 1981; J. F. W.
McOmie, "Protective Groups in Organic Chemistry", pub; Plenum
Press, London, 1973. If the compounds of general formula (XXV) do
not contain any functional groups (apart from a nitro or nitroso
group) which can react with organo-metallic compounds, for example
ester groups, then such a reaction is preferred for the preparation
of compounds of general formula (VII). Disturbing radicals can
possibly be protected and, after the reaction with the
organo-metallic compound, the protective groups are again
removed.
If Q is an N,N-dialkylamino radical and especially a dimethylamino
radical, then the coloured materials of the general formula:
##STR38## in which R.sup.1 and R.sup.2 have the same meanings as
given for general formula (XXVI), L is the radical described in
more detail for general formula (XXV) and Q is a dialkylamino
radical, which are formed as intermediates can be isolated and
converted by alkaline hydrolysis, for example with sodium carbonate
in water/ethanol, into the desired coloured materials of general
formula (VII).
The reaction of nitroso or nitro compounds with organo-lithium or
Grignard compounds is described in Houben-Weyl, Methoden der
organischen Chemie, Vol. 10/1, pp. 1087, 1126; J. Chem. Soc., C,
2119/1971 and leads, depending upon on the excess used, to
hydroxylamine derivatives or to secondary amines and further
products. Surprisingly, we have found that the compounds of general
formulae (XXV) and (XXVI), in the case of the use of equimolar
amounts and subsequent aqueous working up of the reaction mixture,
can be reacted with high yields to give coloured materials of
general formula (VII).
For the amino compounds of general formula (VIII), there are the
following methods of preparation. When L is the radical of general
formula (II), as is defined for compounds of general formula (I),
these amino compounds are known or can be prepared analogously to
the known compounds. Usually, correspondingly substituted
N,N-anilines are used as starting materials which are nitrosated.
Reduction of the nitroso group gives the p-phenylenediamine
derivatives of the general formula L-NH.sub.2 (see J.A.C.S., 73,
3100/1951).
Compounds of general formula (VIII), wherein L is a
pyrazolo-heterocyclic radical of general formula (III), can be
prepared analogously to known methods by converting a compound of
general formula (X), in which L is a pyrazoloheterocyclic radical
of general formula (III), by known methods into the corresponding
amino compound. This can be achieved
a) by reaction with nitric acid or nitric acid in admixture with
sulphuric acid and/or acetic anhydride to give the corresponding
nitro compound or
b) by reaction with nitrous acid to give the corresponding nitroso
compound or
c) by reaction with an aromatic diazonium salt to give the
corresponding arylazo compound and subsequent reduction.
Nitro, nitroso and arylazo radicals, i.e. radicals of the general
formula aryl--N=N-- in which aryl can have the same meanings as
described hereinbefore for aryl radicals and other groups
containing such radicals, can be converted into amino groups by
reduction with reagents such as zinc in an acid, for example
hydrochloric acid or acetic acid, sodium dithionite, tin in an
acid, for example hydrochloric acid, stannous chloride or by
catalytic hydrogenation, for example in the presence of
palladium-carbon. Such reactions are described in Houben-Weyl,
Methoden der organischen Chemie, Vol. 11/1, p. 341 et seq.
The introduction of nitro, nitroso or arylazo groups starting from
compounds of general formula (X) can take place by nitration with
nitric acid or nitric acid in admixture with concentrated sulphuric
acid or acetic anhydride.
By nitrosation with nitrous acid or by azo coupling with aromatic
diazonium salts, the nitroso group or an arylazo radical can be
introduced. Examples of such reactions are described in
Houben-Weyl, Methoden der organischen Chemie, Vol. 10/1 and
10/3.
If heterocyclic compounds of general formula (X) are present in
which H is not a hydrogen atom but rather a carboxyl,
alkoxycarbonyl or alkylcarbonyl radical, then these can be
converted by hydrolysis with concentrated hydrochloric acid or, in
the case of carboxylic acid, by thermal decarboxylation, into
compounds of general formula (X). This is then followed by the
introduction of an nitro, nitroso or arylazo radical.
Nitrogen atoms which are not on a double bond and in which the
radicals X-Y or Z of general formula (III) occur can optionally be
alkylated or aralkylated. The N-alkylation or N-aralkylation can be
carried out by reaction of the appropriate compounds of general
formula (X) but preferably of those heterocyclic compounds in which
H is not a hydrogen atom but rather nitro, nitroso, alkoxycarbonyl,
acyl or arylazo, with alkylation or aralkylation agents, for
example alkyl or aralkyl halides, dialkyl or diaralkyl sulphates or
arylsulphonic acid alkyl esters or aralkyl esters in the presence
of a base, for example sodium hydride, a tertiary amine, an alkali
metal carbonate or sodium hydroxide, in a solvent, for example
dimethylformamide or an aqueous alcoholic system.
The required starting compounds of general formula (X) or those
compounds corresponding to general formula (X) in which H is
replaced by alkoxycarbonyl or acyl, whereby L is a
pyrazolo-heterocyclic radical of general formula (III), have been
described or can be synthesised analogously to known compounds.
Information regarding the preparation of the heterocyclic systems
is contained in the following publications: G. P. Ellis, "Synthesis
of fused Heterocycles" in "The Chemistry of Heterocyclic
Compounds", E. C. Taylor ed., pub. John Wiley & Sons; P. N.
Preston, "Condensed Imidazoles", in "The Chemistry of Heterocyclic
Compounds", A. Weissberger and E. C. Taylor eds., 1986, pub. John
Wiley & Sons; Adv. of Het. Chem., 36, 343/1984; Chem. Pharm.
Bull., 22, 482/1974; J. Het. Chem., 12, 481/1975; Chem. Pharm.
Bull., 22, 1814/1974; Ann., 660, 104/1962; Chem. Pharm. Bull., 23,
452/1975; J. Het. Chem., 10, 411/1973; J. Chem. Soc. Perkin I,
2047/1977.
The compounds of general formula (I) are outstandingly useful as
hydrolase substrates. Especially preferred for this use are
compounds of general formula (I) as have been described
hereinbefore. Quite especially preferred are compounds of general
formula (I) in which G is an N-acetyl-.beta.-D-glucosaminidyl
radical for use as substrate for the enzyme
N-acetyl-.beta.-D-glucosaminidase (NAGase).
For the carrying out of the process according to the present
invention for the colorimetric determination of a hydrolase, a
compound of general formula (I) is reacted, as enzyme substrate,
with the enzyme to be determined, the leuko coloured material
resulting from the substrate cleavage is oxidised and the coloured
material resulting therefrom is determined visually or
photometrically. It represents a measure for the amount of enzyme
to be determined.
For the oxidation of the leuko coloured material of general formula
(V) first resulting by the action of the hydrolase, there can be
used any oxidation agent which does not influence the activity of
the enzyme to be determined and which is strong enough to oxidise
the resulting leuko coloured material. As a rule, as oxidation
agent there is used potassium ferricyanide, perborate, bilirubin
oxidase, peroxidase/hydrogen peroxide or, preferably, iodate.
As can be gathered from the above remarks, a reaction of the
substrate by the enzyme in the presence of the oxidation agent is
preferred. However, it is also possible to carry out the hydrolase
reaction and the oxidation separately. However, according to the
latter procedure, a kinetic determination is not possible but only
an end point determination.
It is self-evident that a buffer system adjusted to the enzyme must
be present for carrying out the determination process. Which buffer
is the most suitable is known. For example, the determination
process according to the present invention for
N-acetyl-.beta.-D-glucosaminidase as hydrolase is carried out at a
pH value of from 3.5 to 7.0 and preferably of from 4.0 to 6.5.
The diagnostic agent for the determination of a hydrolase according
to the present invention contains, besides one or more substrates
of general formula (I) according to the present invention, an
appropriate buffer system, as well as possibly further appropriate
additives conventional for such reagents, for example further
adjuvant enzymes, stabilizers and the like. The oxidation agent
necessary for the formation of the coloured material after cleavage
of the enzyme substrate can be present together with the other
substances necessary for the determination process or can be
separate therefrom. The reagent according to the present invention
can be present in the form of a solution, as a lyophilisate, as a
powder mixture, as a reagent tablet or on an appropriate carrier
material, whereby the reagent components can be worked up to give a
diagnostic agent together, separately or, depending upon
compatibility and expediency, combined with one another.
The diagnostic agent according to the present invention in the form
of a solution preferably contains all the reagents required for the
test. As solvent, there can be used water or mixtures of water with
a water-soluble organic solvent, for example methanol, ethanol,
acetone or dimethylformamide. For reasons of storage stability, it
can be advantageous to divide the reagents required for the test
into two or more solutions which are first mixed together in the
case of carrying out the actual investigation.
For the preparation of the diagnostic agent in the form of a
lyophilisate with a total weight of, in each case, about 5 to 20
mg. and preferably of about 10 mg., a solution is dried which
contains all reagents needed for the test, as well as conventional
structure formers, for example polyvinylpyrrolidone and possibly
further filling materials, for example mannitol, sorbitol or
xylitol. However, the oxidation agent can be present in the
diagnostic agent separated from the other components. This can be
achieved by separate lyophilisation or by admixture of the
undissolved oxidation agent.
A reagent in the form of a powder mixture or reagent tablet can be
prepared by mixing the components of the test with conventional
galenical additives and granulating. Additives of this kind
include, for example, sugar alcohols, such as mannitol, sorbitol or
xylitol, or other soluble insert compounds, such as polyethylene
glycol or polyvinylpyrrolidone. In general, the powder mixture or
reagent tablet has an end weight of about 30 to 200 mg. and
preferably of 50 to 80 mg.
Solid reagent mixtures, such as lyophilisates, powder mixtures or
tablets, are, before use, dissolved in water or some other
appropriate solvent and the reagent solution(s) thus prepared.
After mixing the sample with a sufficient amount of the reagent
mixture, the resultant colour is measured on a photometer and the
particular enzyme concentration calculated via the molar extinction
coefficient and the added volumes of reagent or sample. Not only
kinetic but also end point measurements are possible.
For the production of the reagent in the form of a test strip, an
absorbent carrier, preferably filter paper, cellulose or synthetic
material fibre fleece, is impregnated with solutions of the
necessary reagents conventionally used for the production of test
strips in a ready volatile solvent, for example water, methanol,
ethanol or acetone. This can take place in one impregnation step.
However, it is often desirable to carry out the impregnation in
several steps, whereby solutions are used which, in each case,
contain a part of the components of the reagent. Thus, for example,
in a first step, impregnation can be carried out with an aqueous
solution which contains the oxidation agent, the buffer and
possibly other water-soluble additives and then, in a second step,
with a solution which contains the hydrolase substrate. The
finished test strips can be used as such or can be stuck in known
manner on to handles or preferably sealed between synthetic
materials and fine meshes according to Federal Republic of Germany
Patent Specification No. 21 18 455, U.S. Pat. No. 3,802,842.
As preferred diagnostic agent according to the present invention,
there is produced a test carrier according to FIG. 5 of the
accompanying drawings. This test carrier consists of an absorbent
carrier material, such as paper (1), containing a buffer and
hydrolase substrate, arranged hereon, in planar contact with the
absorbent carrier (1), a mesh (2) containing the oxidation agent,
for example a mesh of polymeric material and a covering mesh (3)
fixing (1) and (2) on a handle of plastic film (4) in order to
simplify handling. This covering mesh (3) can also consist of a
polymeric material. The covering mesh (3) itself is fixed by means
of a melt adhesive (5) on to the film (4).
For carrying out a hydrolase determination, the sample to be
investigated is applied to the covering mesh (3) of the test
carrier according to FIG. 5 or this is dipped into the liquid
sample to be investigated. The sample quickly penetrates through
the covering mesh (3) and, with dissolving of the oxidation agent,
through the mesh (2) into the absorbent material (1) where, in the
case of the presence of the enzyme, enzymatic cleavage of the
there-present enzyme substrate and oxidation of the intermediate
formed leuko coloured material to the determinable coloured
material takes place.
A semi-quantitative determination is possible by associating the
resultant colour with a comparison colour. A quantitative
evaluation can take place remission photometrically.
As a rule, the compounds of general formula (I) are sufficiently
storage-stable for use in diagnostic agents according to the
present invention. However, we have ascertained that the stability
of the hydrolase substrates according to the present invention can
be still further increased when they are present together with
compounds of the general formula:
wherein A.sub.r is an aryl radical optionally substituted by alkyl,
alkoxy or halogen.
In this connection, alkyl, alkoxy, halogen and aryl have the same
meanings as given hereinbefore for the substituents in general
formula (I). Especially advantageous compounds of general formula
(XII) are p-methyl-, o-methoxy-, m-methoxy-, p-methoxy, o-chloro-,
m-chloro-, o-chloro-, o-methyl, m-methyl- and unsubstituted
phenyl-semicarbazides, as well as naphthyl semicarbazide.
The compounds of general formula (XII) have proved to be quite
especially advantageous for increasing the storage stability of
N-acetylglucosaminides according to the present invention.
In the scope of the present invention, the compounds of general
formula (I) display many advantages. In particular, it is of
considerable advantage that the substrates according to the present
invention are colourless and, in the case of carrying out the
process according to the present invention, coloured substances are
formed, the red or blue colour of which can clearly be ascertained.
The large wavelength shift, i.e. the difference of the maximum
absorption wavelengths of substrate and coloured material formed,
makes possible very sensitive determinations. Such determinations
are possible especially in biological fluids, for example plasma,
serum and, in particular, urine since the resultant colour differs
very clearly from the sample material. The coloured materials
formed after cleavage of the substrates according to the present
invention are formed over a very wide pH range. Above all, it is
important that, according to the present invention, coloured
materials are formed not only in the neutral and basic pH region
but also already at pH values in the rather acidic range, i.e. from
pH 3.5, so that no rebuffering is necessary in the case of a
determination of "acidic" enzymes, i.e. those enzymes which display
their activity maximum in the acidic ph range. This makes the
compounds of general formula (I) according to the present invention
especially appropriate substrates for "acidic" enzymes, for example
NAGase. The carrying out of the hydrolase determination according
to the present invention at the pH optimum of the enzymes with
non-coloured substrates makes possible the rapid detection even of
low enzyme concentrations. Finally, the substrates according to the
present invention are suitable not only for wet chemical
determinations in solutions but also for dry chemical
determinations on test carriers. The evaluation can take place
visually or photometrically in transmission or remission.
The following Examples are given for the purpose of illustrating
the present invention:
EXAMPLE 1
4-(4'-Dimethylaminophenylamino)-phenyl-2-acetamido-2-deoxy-.beta.-D-glycopy
ranoside. ##STR39##
To prepare the starting material N,N-Dimethylindoaniline (Phenol
Blue III)-A solution of 0.075 mole of phenol, 4 g (0.1 mole) of
sodium hydroxide, and 8 g (0.06 mole) of sodium acetate in 250 c.c
of water was stirred mechanically in a salt-ice bath in such a way
as to maintain a temperature of 0.degree. to 5.degree. during the
reaction. Two dropping funnels were put in place, one containing
100 cc (0.1 mole) of 5% sodium hypochlorite, the other a solution
of 0.05 mole of p-aminodimethylaniline hydrochloride in 100 cc of
water. The two solutions were then added simultaneously in the
course of forty-five to sixty minutes. The solution became blue
after the addition of a few drops of the reagents and the dye soon
began to separate. Stirring was then continued for fifteen minutes
after completion of the addition and the product was then collected
and washed several times with water; the dark blue filtrate,
probably containing phenol-indophenol formed by alkaline hydrolysis
of the product was discarded. The crude dye was crystallized from
ethyl acetate, giving fine, intense violet needles, m.p.
161.degree. [as reported by Gnelm and Bots J. Prakt. Chem., 69:162
(1904)] Yield 7.2 g (63%). This is generally the method of Gnelm
and Bots, Supra [see also Heller Ann 392:16 (1912) and Cohen and
Phillips, Suppl. No. 74 U.S. Pub. Health Reports 1929]. 1.1 6 g
N,N-Dimethylindoaniline (J.A.C.S., 61, 376/1939, see above) are
added to a solution of 7 g. sodium hydroxide in 250 ml. water.
While stirring vigorously, sodium dithionite is added portionwise
thereto until the blue colour has disappeared. The solution is
filtered, the filtrate is cooled to about 5.degree. C. and mixed
dropwise with glacial acetic acid, until the precipitate has come
out completely. The precipitate is filtered off and dissolved in 25
ml. concentrated hydrochloric acid. The solution is treated with
carbon, filtered and evaporated. The residue is recrystallised from
ethanol. There are obtained 3.1 g. (51% of theory)
4-dimethylamino-4'-hydroxydiphenylamine. 1.2 2.6 g. of the leuko
coloured material obtained in 1.1, 8.83 g.
2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-.alpha.-D-glucosyl chloride
and 4.21 g. benzyltriethylammonium bromide are added to a mixture
of 125 ml. chloroform and 125 ml. water. The mixture is vigorously
stirred, mixed with 8.7 g. potassium carbonate and boiled under
reflux for 6 hours, whereby after 3 hours, 4.4 g. of the halogenase
and 4.3 g. potassium carbonate are added thereto. The chloroform
phase is separated off, dried with anhydrous sodium sulphate and
evaporated.
The residue is chromatographed on silica gel with ethyl acetate.
The product-containing fractions are evaporated. There are obtained
500 mg. (9% of theory) of the protected sugar derivative of the
following formula: ##STR40## 1.3 460 mg. of the protected sugar
derivative obtained in 1.2 are dissolved in 10 ml. methanol, mixed
with 0.9 g. sodium bicarbonate and vigorously stirred for 3 hours
at ambient temperature. The reaction mixture is filtered, the
filtrate is evaporated and the residue is chromatographed on silica
gel with ethyl acetate/methanol (8:2 v/v). The product-containing
fractions are combined and evaporated. The residue is taken up in a
little methanol and the solution is mixed with diethyl ether. The
precipitate thus obtained is filtered off with suction and washed
with diethyl ether. There is obtained 0.34 g. (96% theory) of the
title compound; m.p. 207.degree.-209.degree. C. (decomp.).
EXAMPLE 2
4-((N-acetyl-4'-dimethylaminophenyl)-amino)-phenyl-2-acetamido-2-deoxy-.bet
a.-D-glucopyranoside. ##STR41##
0.3 g. of the protected glycoside obtained in Example 1.2 is mixed
with 5 ml. acetic anhydride, heated to 80.degree. C. and maintained
at this temperature for 2 hours. The reaction mixture is evaporated
and the residue is mixed with diethyl ether. The precipitate
obtained is filtered off and, for the splitting off of the
protective groups, is treated with sodium bicarbonate, analogously
to Example 1.3, in methanol. The crude product is chromatographed
over silica gel with toluene/ethyl acetate/methanol (1:1:1 v/c/v).
There is obtained 0.1 g. (47% of theory) of the title compound;
m.p. 142.degree.-144.degree. C. R.sub.f (silica gel, toluene/ethyl
acetate/methanol 1:1:1 v/v/v)=0.37.
EXAMPLE 3
4-((4'-Dimethylaminophenyl-N-trifluoroacetyl)-amino)-phenyl-2-acetamido-2-d
eoxy-.beta.-D-glucopyranoside. ##STR42##
Analogously to Example 2, with the use of trifluoroacetic anhydride
instead of acetic anhydride with a reaction temperature of
0.degree. C., there is obtained the title compound; m.p.
191.degree.-198.degree. C. R.sub.f (silica gel, toluene/ethyl
acetate/methanol 2:1:1 v/v/v)=0.35.
EXAMPLE 4
Analogously to Examples 1.2 and 1.3, from the corresponding leuko
coloured materials and
2-acetamido-3,4,6-tri-0-acetyl-2-deoxy-.alpha.-D-glucosyl chloride,
there are obtained the compounds set out in the following Table
1.
TABLE 1
__________________________________________________________________________
leuko coloured material com- m.p. ponent; prepn. No. structure
.degree.C. R.sub.f see
__________________________________________________________________________
4a ##STR43## 180-188 0.56.sup.2) 4.1 4b ##STR44## 197 0.27.sup.1)
4.2 4c ##STR45## 205 0.16.sup.1) 4.2 4d ##STR46## 194 0.16.sup.1)
4.2 4e ##STR47## 188-191 0.35.sup.2) 4.3 4f ##STR48## 195
0.35.sup.1) 4.2 4g ##STR49## 210-212 0.5.sup.2) 4.2 4h ##STR50##
143 0.27.sup.1) 4.4 4i ##STR51## 212 0.40.sup.1) 4.5
__________________________________________________________________________
.sup.1) silica gel, toluene/ethyl acetate/methanol, 2:1:1 v/v/v
.sup.2) silica gel, toluene/ethyl acetate/methanol, 1:1:1 v/v/v
Preparation of the leuko coloured material components. 4.1.
4-Dimethylamino-2'-chloro-4'-hydroxydiphenylamine.
A solution of 7.7 ml. m-chlorophenol, 4 g. sodium hydroxide and 8
g. sodium acetate in 200 ml. water, cooled to 0.degree. to
-5.degree. C., is mixed, with good stirring, simultaneously from
two dropping funnels with, in each case, a solution of 10.45 g.
N,N-dimethylphenylenediamine hydrochloride in 100 ml. water and 125
ml. of a 4% sodium hypochlorite solution in the course of 45 to 60
minutes. The reaction mixture is subsequently stirred for 15
minutes and the precipitate is filtered off and washed several
times with water. The residue is dissolved in a mixture of 50 ml.
2N aqueous sodium hydroxide solution, 30 ml. water and 20 ml.
methanol and reduced at a temperature of about 40.degree. to
45.degree. C. (bath temperature) to the leuko coloured material by
the addition of about 10 to 20 g. sodium dithionite. The reaction
mixture is adjusted to pH 6 by the addition of glacial acetic acid
and the precipitate obtained is filtered off with suction. For
purification, the product is chromatographed over silica gel with
ethyl acetate/ligroin (8:2 v/v). There are obtained 3.5 g. of the
title compound.
4.2 The leuko coloured materials 4-dimethylamino-2',
5'-dichloro-4'-hydroxydiphenylamine,
4-morpholino-4'-hydroxydiphenylamine,
4-dimethylamino-3'-chloro-4'-hydroxydiphenylamine,
4-morpholino-2'-chloro-4'-hydroxydiphenylamine and
4-morpholino-2'-fluoro-4'-hydroxydiphenylamine are obtained
analogously to 4.1 by the oxidative coupling of the appropriate
phenylenediamine with the correspondingly substituted phenol
4.3 4-Hydroxyphenyl-2-methylpyrazolo[1,5-a]pyridin-3-yl-amine.
4.3.1 7 g. 3-Acetyl-2-methylpyrazolo[1,5-a]pyridine are dissolved
in 140 ml. 6N hydrochloric acid and mixed dropwise at 0.degree. C.
with a solution of 5.52 g. sodium nitrite in water. After 2 hours,
the ice-bath used for cooling is removed, the reaction mixture is
left to stand overnight at ambient temperature and then adjusted to
pH 9. The precipitated nitroso compound (6.4 g.) is filtered off
with suction and dissolved in about 150 ml. 2N hydrochloric acid.
The nitroso compound is reduced with stannous chloride analogously
to Example 4.4.1.2. The crude product is chromatographed on silica
gel with ethyl acetate. The product-containing fractions are
evaporated, the residue is dissolved in ethanol and mixed with
ethanolic hydrochloric acid. The precipitate which comes put after
some time is filtered off with suction and dried. There are
obtained 3.1 g. (45% of theory)
3-amino-2-methylpyrazolo[1,5-a]pyridine hydrochloride; m.p.
>275.degree. C.; R.sub.f (silica gel, ethyl
acetate/acetone/glacial acetic acid/water 50:25:12.5:12.5
v/v/v/v)=0.6
4.3.2 2.82 g. Phenol are dissolved in 75 ml. pyridine and the
solution is mixed with 450 ml. water. A solution of 5.5 g.
3-amino-2-methylpyrazolo[1,5-a]pyridine hydrochloride from 4.3.1 in
150 ml. water is then added thereto and the reaction mixture is
subsequently mixed, while stirring, with a solution of 78 g.
potassium ferricyanide in 450 ml. water. The precipitated
blue-coloured material is filtered off with suction, washed with
water and dried. Yield 4.85 g.
TLC (silica gel, ethyl acetate/methylene chloride 1:1 v/v): R.sub.f
=0.5.
4.3.2.1 Alternative synthesis of the coloured material
N-(2-methylpyrazolo[1,5-a]pyridin-3-yl)-quinonimine.
From 7.7 g. 4-bromodimethylaniline and 1.4 g. magnesium turnings in
60 ml. dry tetrahydrofuran is prepared the corresponding Grignard
compound (see J. Chem. Soc., 465/1961). The reaction solution is
sucked off with a syringe and added dropwise to a solution, cooled
to -15.degree. C., of 3.05 g.
3-nitroso-2-methylpyrazolo[1,5-a]pyridine (prepared according to
Example 4.3.1) in 60 ml. tetrahydrofuran. After warming, the
reaction mixture is poured into 100 ml. 2N acetic acid. The
tetrahydrofuran is stripped off on a rotary evaporator. The
reaction mixture is mixed with 100 ml. ethanol and an excess of
sodium carbonate until the reaction is alkaline. For the oxidation
of the resultant leuko coloured material, there is added thereto a
total of 40 g. potassium ferricyanide and the reaction mixture is
then stirred at ambient temperature. After hydrolysis is complete,
the reaction mixture is extracted with ethyl acetate. The organic
phase is evaporated and the residue is chromatographed on silica
gel with the elution agent ethyl acetate/ligroin (1:1 v/v). There
are obtained 4.4 g. (98% of theory, referred to the nitroso
compound) of the coloured material. R.sub.f (silica gel, methylene
chloride/ethyl acetate (1:1 v/v))+0.6. 4.3.3. For reduction to the
leuko coloured material, the coloured material is dissolved in 200
ml. ethyl acetate and the solution is mixed with about 100 ml.
saturated aqueous sodium carbonate solution. Until decolorised, the
solution is vigorously shaken with sodium dithionite. The organic
phase is separated off, dried and concentrated to a small volume.
This is then mixed with ligroin and the precipitate obtained is
filtered off, washed with ligroin and subsequently dried. There are
obtained 4.45 g. of the title compound which is pure enough for
further working up. A purification is possible by chromatography
over silica gel with methylene chloride/methanol (98:2 v/v).
R.sub.f (silica gel, ethyl acetate/methylene chloride (1:1
v/v))=0.6.
R.sub.f (silica gel, methylene chloride/methanol (95:5
v/v))=0.3.
4.4 4-Hydroxyphenyl-pyrazolo[1,5-a]pyridin-3-ylamine.
4.4.1 2 g. Pyrazolo[1,5-a]pyridine are dissolved in 30 ml. 6N
hydrochloric acid, the solution is cooled to 0.degree. C. and a
solution of 6.9 g. sodium nitrite in 30 ml. water is slowly added
dropwise thereto. After 1 hour, the nitrosation is complete. About
100 ml. water are added thereto, followed by repeated extraction
with ethyl acetate. The organic phase is dried and evaporated.
There are obtained 9.6 g. 3-nitrosopyrazolo[1,5-a]pyridine. 4.4.2 9
g. of the nitroso compound obtained according to 4.4.1 are
introduced into a solution of 22 g. stannous chloride dihydrate in
180 ml. concentrated hydrochloric acid. The reaction mixture is
stirred for 1 hour at ambient temperature and, for the completion
of the reduction, mixed with 8 g. stannous chloride dihydrate in 30
ml. concentrated hydrochloric acid. The suspension is poured on to
about 150 g. ice, adjusted with sodium hydroxide to pH 12 and
quickly extracted with ethyl acetate. The ethyl acetate phase is
dried and evaporated. The residue is dissolved in about 350 ml.
diethyl ether and mixed with ethereal hydrochloric acid. The
precipitate obtained is filtered of, washed with diethyl ether and
dried. There are obtained 11.3 g. (100% of theory)
3-aminopyrazolo[1,5-a]pyridine hydrochloride; m.p.
228.degree.-232.degree. C. R.sub.f (silica gel, ethyl
acetate/methanol 9:1 v/v)=0.52. 4.4.3. The leuko coloured material
4-hydroxyphenyl-pyrazolo[1,5-a]pyridin-3-ylamine is obtained
analogously to 4.3.2 and 4.3.3 with the use of
3-aminopyrazolo[1,5-a]pyridine as starting material. R.sub.f
(silica gel, ethyl acetate/diethyl ether 1:1 v/v)=0.68.
4.5
4-Hydroxyphenyl-2-methylthiopyrazolo[1,5-a]pyridin-3-ylamine.
With 2,2-bis(methylthio)-1-nitroethylene (II) a reaction with Ia
afforded 2-methylthio-1-nitropyrazolo [1,5-a]pyridine (III) as
yellow needles, m.p 224.6.degree. in 41% yield. The product where R
is H. ##STR52##
4.5.1 4 g. 2-Methylthio-3-nitropyrazolo[1,5-a]pyridine (see
Heterocycles, 6, 379 (1977), Chem. Pharm. Bull. 25 1528/1977) are
dissolved in 200 ml. concentrated hydrochloric acid and mixed with
20 g. stannous chloride dihydrate. After 1 hour, 15 g. stannous
chloride dihydrate, 15 ml. concentrated hydrochloric acid and 200
ml. water are again added thereto. After a further reaction period
of 1 hour, the reaction mixture is poured on to ice, the yellow
solution is rendered alkaline with sodium hydroxide and extracted
with ethyl acetate. The organic phase is dried and evaporated. The
residue is dissolved in a little ethanol and mixed with ethereal
hydrochloric acid in order to form the hydrochloride. There are
obtained 3.9 g. (95% of theory)
3-amino-2-methylthiopyrazolo[1,5-a]pyridine hydrochloride; m.p.
226.degree. C. R.sub.f (silica gel, methylene chloride/tert.-butyl
methyl ether 2:8 v/v)=0.66.
4.5.2 The leuko coloured material
4-hydroxyphenyl-2-methylthiopyrazolo[1,5-a]pyridin-3-ylamine is
obtained analogously to 4.3.2 and 4.3.3 with the use of
3-amino-2-methylthiopyrazolo[1,5-a]pyridine. R.sub.f (silica gel,
ethyl acetate/ligroin 1:1 v/v)=0.47.
EXAMPLE 5
4-((R,S-2-(1-Hydroxyethyl)-pyrazolo[1,5,a]pyridin-3-yl)amino)-phenyl-2-acet
amido-2-deoxy-.beta.-D-glucopyranoside. ##STR53##
5.1
4-Hydroxyphenyl-2-(1-hydroxyethylpyrazolo[1,5-a]pyridin-3-yl)-amine.
25 g. N-Aminopyridine hydrochloride are dissolved in 250 ml. dry
dimethylformamide and mixed, while stirring, with 17.5 g. potassium
carbonate. Subsequently, while stirring, 20.2 g.
R,S-2-hydroxy-5-oxohex-3-yne (J. Chem. Soc. Perkin I, 1908/1976)
are added dropwise thereto. The reaction mixture thereby warms up
and is left to stand overnight. After the addition of 1.25 liters
water, the reaction mixture is extracted several times with ethyl
acetate. The combined extracts are dried and evaporated. The
remaining oil is diluted with some diethyl ether. The crystals
which precipitate out after some time are filtered off with
suction. There are obtained 9.8 g. (42% of theory)
R,S-3-acetyl-2-(1-hydroxyethyl)-pyrazolo[1,5-a]pyridine (R.sub.f
(silica gel, methylene chloride/ethyl acetate 1:1 v/v =0.35 ) which
is nitrosated analogously to Example 4.3.1. The nitroso compound is
suspended in ethanol and mixed with 2 g. palladium on carbon. The
reaction mixture is heated to 80.degree. C. and 2.5 ml. hydrazine
hydrate are added portionwise thereto. After 10 minutes, the
palladium-carbon is filtered off and the filtrate is evaporated.
The residue is dissolved in ethanol and mixed with ethanolic
hydrochloric acid. There are obtained 6.8 g.
R,S-3-amino-2-(1-hydroxyethyl)-pyrazolo[1,5-a]pyridine
hydrochloride in the form of crystals, which contain 1.8 mole
hydrogen chloride and melt at 229.degree.-231.degree. C. R.sub.f
(silica gel, ethyl acetate/methanol 3:1 v/v)=0.65.
Analogously to Example 4.3.2, 3.08 g. phenol are reacted with the
amino compound obtained and the blue-coloured material is reduced
analogously to Example 4.3.3. The crude product is chromatographed
over silica gel with ethyl acetate/methylene chloride (1:1 v/v) as
elution agent. There are obtained 3.4 g. of the title compound.
R.sub.f (silica gel, ethyl acetate/methylene chloride 1:1 v/v)=0.2
5.2 Analogously to Example 1.2 and 1.3, from the leuko coloured
material from 5.1 and
2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-.alpha.-D-glucosyl chloride,
there is obtained the title compound. R.sub.f (silica gel,
toluene/ethyl acetate/methanol 1:1:1 v/v/v)=0.25; m.p.
165.degree.-170.degree. C. (decomp.).
To prepare Hex-3-yne-2,5-dioxe: Chromium trioxide (100 g) in
concentrated sulphuric acid (90 ml) and water (550 ml) was added
with vigorous stirring over 2 hours to hex-3-yne-2,5-diol (28 g)
and 5-hydroxyhex-3-yn-2-one (28 g) in acetone (400 ml) at
-5.degree. to 0.degree.. After 1 hour, water (150 ml) was added and
the solution extracted with ether. The extract was washed (aqueous
NaHCO.sub.3), dried (MgSO.sub.4) and distilled to give crude
hex-3-yne-2,5-dione (3-6 g), B.P. 50.degree. (bath) at 0.15 m Hg
and 5-hydroxy-hex-3-yne-2-one (B.P 50.degree.-75.degree. at 0.2 mm
Hg) 23 g.
EXAMPLE 6
Wet chemical determination of N-acetyl-.beta.-D-glucosaminidase
(.beta.-NAGase).
900 .mu.l. of a solution of 4 mM substrate in 200 mM citrate buffer
(pH 5.) are placed in a cuvette. To this are pipetted:
1. 100 .mu.l. of a 220 mM potassium iodiate solution and 2. 100
.mu.l. of a solution containing .beta.-NAGase, mixed and the
kinetics measured at a wavelength corresponding to the substrate.
Calibration curves according to FIGS. 1-4 of the accompanying
drawings can be obtained with solutions containing known
concentrations of NAGase. 6.1. The substrate from Example 4e)
gives, at .lambda..sub.max 560 nm, a calibration curve for the
determination of .beta.-NAGase according to FIG. 1 of the
accompanying drawings. In the case of the presence of
.beta.-NAGase, the colour of the substrate changes from colourless
to blue-violet. 6.2. The substrate from Example 4g) gives, at
.lambda..sub.max 573 nm, a calibration curve for the determinations
of .beta.-NAGase according to FIG. 2 of the accompanying drawings.
In the case of the presence of .beta.-NAGase, the colour of the
substrate changes from colourless to blue-violet. 6.3. The
substrate from Example 4h) gives, at .lambda..sub.max 525 nm, a
calibration curve for the determination of .beta.-NAGase according
to FIG. 3 of the accompanying drawings. In the case of the presence
of .beta.-NAGase, the colour of the substrate changes from
colourless to red-violet. 6.4. The substrate from Example 5 gives,
at .lambda..sub.max 565 nm, a calibration curve for the
determination of .beta.-NAGase according to FIG. 4 of the
accompanying drawings. In the case of the presence of
.beta.-NAGase, the colour of the substrate changes from colourless
to blue-violet.
EXAMPLE 7
Test strips for the determination of .beta.-NAGase.
a) A filter paper of the firm Schleicher & Schull (23 SL) is
successively impregnated with the following solutions and
dried:
______________________________________ 1. citrate buffer 200
mMole/liter, pH 5 potassium iodate 20 mMole/liter 2. indicator 10
mMole/liter ______________________________________
As indicator, there is used:
.alpha.) the compound of Example 4e)
.beta.) the compound of Example 4h)
When the so-produced test strip is dipped into a solution
containing NAGase, then, after about 5 minutes, above about 10
U/liter .beta.-NAGase (the activity is determined according to the
present reference method with
sulphophthaleinyl-N-acetyl-.beta.-D-glucosaminide as enzyme
substrate) there is obtained a coloration of the test strip from
colourless to blue when the compound of Example 4e) is used and
from colourless to red in the case of using the compound of Example
4h). Higher enzyme activities lead to more intensive
coloration.
b) A comparable result is achieved when the test strip from a)
above is so modified that the reagent paper is impregnated as above
but with a solution without iodate. The oxidation agent is
impregnated from a 40 mMole/liter aqueous sodium iodate solution on
to a nylon mesh (NY75HC of the firm Zurcher Peuteltuchfabrik,
Zurich, Switzerland) with a filament thickness of 60 .mu.m. From
the paper (1) containing buffer and indicator, the mesh (2)
containing the oxidation agent, a covering mesh (3) of the same
material as mesh (2) and a stiff plastic film (4) of polystyrene is
produced a test carrier according to FIG. 5 of the accompanying
drawings. For this purpose, the paper (1) and the mesh (2) are cut
up into 6 mm..times.6 mm. sized pieces and fixed with a 12
mm..times.6 mm. sized piece of covering mesh (3) by means of melt
adhesive (5) on to a 100 mm..times.6 mm. sized piece of plastic
film (4).
Upon dipping such a test strip into a solution containing
.beta.-NAGase, a coloration takes place from colourless to blue for
the compound 4e) and from colourless for the compound 4h). Higher
enzyme concentrations lead to more intensive coloration.
EXAMPLE 8
N-(p-toluenesulphonyl)-L-alanine-(4-((2-methylpyrazolo-[1,5-a]pyridin-3-yl)
-amino)-phenyl ester. ##STR54##
0.48 g. of the leuko coloured material obtained in Example 4.3 is
dissolved in 8 ml. pyridine and, while stirring, mixed dropwise
with a solution of p-toluene-sulphonyl chloride in 12 ml.
chloroform. After briefly after-stirring, the reaction mixture is
mixed with water and the organic phase is separated. The organic
phase is well washed with water, separated and dried. The residue
is chromatographed with the elution agent water/methanol on the
adsorber resin HP 20SS (firm Mitsubishi). The product-containing
fractions are evaporated and the residue is crystallised from
diethyl ether/hexane. There is obtained 0.54 g. of the title
compound; m.p. 68.degree.-78.degree. C. (decomp.).
R.sub.f =0.7 (silica gel, ethyl acetate/methylene chloride, 1:1
v/v).
EXAMPLE 9
Phosphoric acid
mono-(4-((2-methyl-pyrazolo[1,5-a]-pyridin-3-yl)-amino)-phenyl
ester, pyridinium salt. ##STR55##
0.35 g. of the leuko coloured material obtained in Example 4.3 is
dissolved in 3 ml. pyridine. The solution is cooled to -15.degree.
C. and, while stirring, mixed dropwise with a solution of 0.15 ml.
phosphorus oxychloride in 2.5 ml. pyridine. The reaction mixture is
stirred for 30 minutes at -15.degree. C. and for 2 hours at ambient
temperature, mixed with ice, acidified with 2N sulphuric acid to pH
2 and left to stand overnight in a refrigerator. The precipitate
obtained is filtered off and the filtrate is passed over a column
containing the adsorber resin HP 20SS (Mitsubishi). The product is
eluted with water. The product-containing fractions are combined,
evaporated and the residue crystallised from methanol/diethyl
ether. There is obtained 0.06 g. of the title compound: m.p.
158.degree.-161.degree. C. (decomp.).
EXAMPLE 10
4-(2,4-Dimethylpyrazolo[1,5-a]imidazol-3-yl)-amino)phenyl-2-acetamido-2-deo
xy-.beta.-D-glucopyranoside. ##STR56## 10.1 1.92 g.
1,2-dimethylimidazole are dissolved in 10 ml. methylene chloride
and, while cooling with ice, mixed with a solution of
O-p-toluenesulphonyl hydroxylamine, which was obtained by the
reaction of 15.4 g. O-p-toluene-sulphonyl acethydroxamic acid ethyl
ester with 118 ml. 60% aqueous perchloric acid. The reaction
mixture is stirred for 3 hours at ambient temperature. The
precipitate obtained is filtered off and washed with diethyl ether.
There are obtained 5 g. (88% of theory)
N-amino-1,2-dimethylimidazolium p-toluenesulphonate.
10.2 5 g. of the product obtained in 10.1 are heated with 5.4 g.
sodium acetate and 125 ml. acetic anhydride for 1 hour at a bath
temperature of 140.degree. C. The reaction mixture is evaporated in
a vacuum and the residue is taken up in water. The pH value is
adjusted to 9 to 10 and extracted with methylene chloride. The
organic phase is dried and evaporated. The residue is
chromatographed on silica gel with ethyl acetate. There is obtained
0.45 g. (11% of theory)
3-acetyl-2,4-dimethylpyrazolo[1,5-a]imidazole; m.p.
165.degree.-167.degree. C.
10.3 The compound obtained in 10 is nitrosated analogously to
Example 4.3.1. There is obtained 0.35 g. (94% of theory) of the
corresponding nitroso compound; m.p. 179.degree.-183.degree. C.
This is dissolved in 100 ml. dilute aqueous sodium bicarbonate
solution and reduced with sodium dithionite. The reaction mixture
is evaporated and digested with ethanol. The ethanol solution is
evaporated and the product is precipitated out by the addition of
ethereal hydrochloric acid. There is obtained 0.3 g. (80% of
theory) of the amino compound; m.p. 199.degree.-204.degree. C.
(decomp.). TLC (silica gel, ethyl acetate/acetone/glacial acetic
acid/water 50:25:12.5:12.5 v/v/v/v): R.sub.f =0.3
10.4 Analogously to Example 4.3.2, the above-obtained amino
compound is reacted with potassium ferricyanide and phenol and the
coloured material obtained is reduced analogously to Example 4.3.3.
R.sub.f of the leuko coloured material obtained: silica gel,
toluene/ethyl acetate/methanol 2:1:1 v/v/v=0.5.
10.5 0.46 g. of the above-obtained leuko coloured material is
dissolved in 40 ml. methylene chloride and, while cooling with ice,
mixed first with 0.65 ml. diisopropylethylamine and then dropwise
with 0.51 ml. trifluoroacetic acid anhydride. After 1 hour, there
are again successively added thereto 0.35 ml. diisopropylethylamine
and 0.25 ml. trifluoroacetic acid anhydride. After stirring for 30
minutes, the reaction mixture is evaporated and the residue is
chromatographed over silica gel with methylene chloride/ethyl
acetate (1:1 v/v). There is obtained 0.4 g.
N-(2,4-dimethylpyrazolo[1,5-a]imidazol-3-yl)-N-(4-hydroxyphenyl)trifluoroa
cetamide. R.sub.f =0.3 (silica gel, methylene chloride/ethyl
acetate 1:1 v/v).
10.6 The above-obtained phenol derivative is glycosidated
analogously to Example 1.2 with
2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-.alpha.-D-glucosyl chloride.
For the splitting off of the protective groups, including the
trifluoroacetyl radical, the product purified by chromatography on
silica gel (methylene chloride/ethyl acetate 1:1 v/v) is dissolved
in methanol and the solution is mixed with a large excess of
anhydrous sodium carbonate. The mixture is stirred for 1.5 hours at
45.degree. C., the salts are filtered off with suction, the residue
is washed with some methanol and the filtrate is concentrated to
about 20 ml. This solution is applied to a column containing the
adsorber resin HP 20SS (Mitsubishi) and eluted with a stepped
gradient of methanol/water (1:9 to 1:1 v/v). There is obtained the
title compound; m.p. 112.degree.-115.degree. C. (decomp.). R.sub.f
=0.2 (silica gel, toluene/ethyl acetate/methanol 1:1:1 v/v/v).
EXAMPLE 11
4-((2,4-Dimethylpyrazolo[1,5-a]-imidazol-3-yl)-N-trifluoroacetyl)-amino)-ph
enyl-2-acetamido-2-deoxy-.beta.-D-glucopyranoside. ##STR57##
When the splitting off of the protective group of the glycoside
obtained in Example 10.6 is carried out with sodium bicarbonate in
methanol analogously to Example 1.3, then only the acetyl radicals
are split off and, after chromatographic purifiication analogously
to Example 10.6, there is obtained the title compound; m.p.
223.degree.-226.degree. C. (decomp.). R.sub.f =0.21 (silica gel,
toluene/ethyl acetate/methanol 1:1:1 v/v/v).
EXAMPLE 12
Analogously to Example 10.5 and 10.6, starting from the appropriate
leuko coloured materials and
2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-.alpha.-D-glucosyl chloride,
there are obtained the compounds set out in the following Table
2:
TABLE 2
__________________________________________________________________________
colored material m.p. component, No. structure .degree.C. R.sub.f
prepn. see
__________________________________________________________________________
12a ##STR58## 198 0.43.sup.2) 12.1. 12b ##STR59## 173 0.24.sup.2)
12.2. 12c ##STR60## 220 (decomp) 0.31.sup.2) 12.3. 12d ##STR61##
193 0.39.sup.2) 12.4. 12e ##STR62## 228 0.37.sup.2) 12.5. 12f
##STR63## 148 (decomp) 0.17.sup.2) 12.6. 12g ##STR64## 248
0.26.sup.2) 12.7. 12h ##STR65## 188-199 (decomp.) 0.4.sup.2) 12.8.
12i ##STR66## 251-254 (decomp.) 0.4.sup.3) 12.8. 12j ##STR67##
271-273 (decomp.) 0.3.sup.2) 12.9. .sup.3) silica gel, ethyl
acetate/acetone/water/glacial acetic acid 50:25:12.5:.2.5 v/v/v/v
12k ##STR68## 227-231 (decomp.) 0.35.sup.2) 12.10. 12l ##STR69##
188 0.43.sup.4) 12.11. 12m ##STR70## 214-216 (decomp.) 0.3.sup.3)
12.12. 12n ##STR71## 185-187 (decomp.) 0.4.sup.2) 12.13. 12o
##STR72## 270-273 (decomp.) 0.4.sup.2) 12.14. 12p ##STR73## 258-260
(decomp.) 0.45.sup.2) 12.14. 12q ##STR74## 239 0.34.sup.2) 12.15.
12r ##STR75## 220 0.27.sup.2) 12.16. 12s ##STR76## 140 (decomp.)
0.38.sup.2) 12.17. 12t ##STR77## 196-200 (decomp.) 0.4.sup.2)
12.17. 12u ##STR78## 221-224 (decomp.) 0.4.sup.2) 12.17. 12v
##STR79## 150 (decomp.) 0.39.sup.2) 12.17. 12w ##STR80## 154
0.30.sup.2) 12.18. 12x ##STR81## 184-185 0.54.sup.2) 12.19. 12y
##STR82## 215-220 (decomp.) 0.25.sup.2) 12.20. 12z ##STR83##
214-216 (decomp.) 0.3.sup.2) 12.20.3. 12aa ##STR84## 162
0.37.sup.2) 12.21.
__________________________________________________________________________
.sup.1) silica gel, toluene/ethyl acetate/methanol, 2:1:1 v/v/v
.sup.2) silica gel, toluene/ethyl acetate/methanol, 1:1:1 v/v/v
.sup.3) silica gel, ethyl acetate/methanol, 1:1 v/v .sup.4) silica
gel, toluene/ethyl acetate/methanol, 1:1:2 v/v/v
Preparation of leuko coloured material components
12.1
4-Hydroxyphenyl-2-methoxypyrazolo[1,5-a]pyridin-3-yl-amine.
12.1.1 3-Amino-2-methoxy-pyrazolo[I,5-a]pyridine hydrochloride.
2-Hydroxypyrazolo[1,5-a]pyridine (I). After a mixture of ethyl
2-pyridylacetate (3.00 g), HAS (0.60 g) and water (3 ml) was
stirred at room temperature for 30 h, it was extracted with
CH.sub.2 CL.sub.2. The aqueous layer was made alkaline with 10%
Na.sub.2 CO.sub.3 to pH 9 and was extracted with CH.sub.2 CL.sub.2.
The organic extracts were combined, and after drying over
MgSO.sub.4 the solvent was evaporated. The residual oil was shaken
with Et.sub.2 O-10% Na.sub.2 CO.sub.3. After the ether-layer was
dried over MgSO.sub.4, the solvent was evaporated to give the
starting ethyl 2-pyridylacetate (2.11 g, 70.5% yield). The pH of
the basic aqueous layer was adjusted to 5 by adding AcOH to
precipitate brown powder (0.36 g, 45.9% yield, based upon the
consumed starting material). Recrystallization from benzene-hexane
gave 2-hydroxypyrazolo[1,5-a]pyridine (0.32 g, 40.7%) mp
127.degree.-128.degree. C., as colorless leaflets. UV
.lambda..sub.max.sup.meOH (log .epsilon.): 232 (4.59), 280.5
(3.09), 310 (3.10). ##STR85## 3000, 1635, 1535, 1258. Found: C,
62.53; H, 4.48; N, 20.98%. Calcd for C.sub.7 H.sub.6 ON.sub.2 :
C,62.68; H, 4.51; N, 20.98%.
2-Methoxypyrazolo[1,5-a]pyridine (V). To a solution of I(134 mg) in
MeOH (5 ml) was added an excess solution of CH.sub.2 N.sub.2 in
ether and the mixture was left to stand in an icebox for a day.
Evaporation of the solvent gave colorless oil (135 mg) which
exhibited a single spot on a TLC plate (R.sub.f =0.89, 5%
MeOH-CHCL.sub.3 silica gel G.F. nach Stahl). Purification by
passing a short column of SiO.sub.2 gave colorless oil (118 mg,
80.5% yield). UV .lambda..sub.max.sup.meOH nm (log .epsilon.): 234
(4.57), 282 (3.30), 307 (3.31). IR .nu..sub.max.sup.CHl.sub.3
cm.sup.-1 : 3000, 1640, 1540, 1360 Found: C, 64.57; H, 5.31; N,
18.71%. Calcd for C.sub.8 H.sub.8 ON.sub.2 : C, 64.85; H, 5.44; N,
18.91%.
1.48 g. 2-methoxypyrazolo[1,5-a]pyridine (see Bull. Chem. Soc.
Jap., 49, 1980/1976 as above) are dissolved, while cooling with
ice, in 20 ml. concentrated nitric acid and mixed dropwise with
10.5 ml. fuming nitric acid. The reaction mixture is stirred for 30
minutes in an ice-bath and for 1 hour at ambient temperature. The
reaction mixture is poured on to ice, the precipitate obtained is
filtered off with suction and washed with water. There is obtained
1 g. (52% of theory) 2-methoxy-3-nitropyrazolo[1,5-a]pyridine; m.p.
213.degree.-216.degree. C.
0.8 g. of the above-obtained nitro compound is suspended in 80 ml.
2N hydrochloric acid and mixed with zinc dust, while stirring
vigorously. After several additions of zinc dust, there is
obtained, after about 1 hour, a clear solution over a bottom
deposit of excess zinc. This is filtered off and the filtrate is
adjusted to pH 7 with sodium hydroxide. The mixture is then
extracted with ethyl acetate, the organic phase is evaporated and
the remaining oil is filtered with ethanol through a short column
of silica gel. The eluate is evaporated, the residue is dissolved
in diethyl ether and mixed with ethereal hydrochloric acid. The
precipitate obtained is filtered off with suction and again
recrystallised from isopropanol. There is obtained 0.33 g. (42% of
theory) of the title compound (12.1.1); m.p.
238.degree.-241.degree. C. TLV (silica gel, acetone/methylene
chloride/glacial acetic acid, 50:45:5 v/v/v): R.sub.f =0.6.
12.1.2 Analogously to Example 4.3.2, from the amino compound
obtained there is obtained, by oxidative coupling with phenol, the
coloured material which is reduced analogously to Example 4.3.3 and
then trifluoroacetylated analogously to Example 10.5.
12.2
4-Hydroxyphenyl-2,5-dimethyl-7-dimethylaminopyrazolo[1,5-a]pyrimidin-3-yla
mine.
12.2.1
3-Amino-2,5-dimethyl-7-dimethylaminopyrazolo[1,5-a]pyrimidine
hydrochloride.
1.6 g. 2,5-dimethyl-7-hydroxypyrazolo[1,5-a]pyrimidine are heated
under reflux for 40 minutes with 16.6 ml. phosphorus oxychloride
and 0.8 ml. N,N-dimethylaniline. The excess phosphorus oxychloride
is distilled off and the residue is poured on to ice. The mixture
is extracted with methylene chloride, the organic phase is washed
with an aqueous solution of sodium carbonate, dried and evaporated.
The residue is dissolved in 28 ml. ethanol and mixed with 2 g. of a
40% solution of dimethylamine in water. The mixture is stirred for
2.5 hours at ambient temperature, evaporated and the residue is
chromatographed over silica gel with ethyl acetate. There is
obtained 0.86 g. (46% of theory)
2,5-dimethyl-7-(N,N-dimethylamino)-pyrazolo[1,5-a]pyrimidine which
is nitrosated analogously to Example 4.3.1.
The nitroso compound obtained (1 g.) is dissolved in 20 ml.
ethanol, mixed with 0.16 g. palladium/carbon and mixed at the
boiling temperature with 0.4 ml. hydrazine hydrate. The reaction
mixture is further boiled under reflux for 15 minutes, filtered
with suction and the filtrate concentrated somewhat. The residue is
dissolved in a little ethanol and mixed with ethereal hydrochloric
acid in order to precipitate out the hydrochloride. There is
obtained 0.8 g.
3-amino-2,5-dimethyl-7-(dimethylaminopyrazolo[1,5-a]pyrimidine
hydrochloride.
R.sub.f =0.54 (silica gel, chloroform/methanol/methyl ethyl
ketone/glacial acetic acid/water 75:35:25:5:8 v/v/v/v/v).
12.2.2. 6.94 g. Phenol are dissolved in 124 m. pyridine and 743 ml.
water. A solution of 11.4 g. of the above-obtained amino compound
in 248 ml. water are added thereto. Finally, one mixes with a
solution of 0.33 g. peroxidase (from horseradish, Boehringer
Mannheim GmbH, Mannheim, Germany) and immediately adds dropwise
thereto 11.23 ml. 30% hydrogen peroxide. There are obtained 12.9 g.
of blue
N-(2,5-dimethyl-6-dimethylaminopyrazolo[1,5-a]pyrimidin-3-yl)-quinonimine.
R.sub.f =0.43 (silica gel, ethyl acetate/methylene chloride 95:5
v/v). The coloured material is reduced analogously to Example 4.3.3
and trifluoroacetylated analogously to Example 10.5. If the
coloured material does not precipitate out, then it is extracted
with ethyl acetate. Under certain circumstances, it is recommended
to work up to the leuko coloured material by the addition of sodium
dithionite and aqueous sodium carbonate solution analogously to
Example 4.3.3.
12.3
4-Hydroxyphenyl-2,4,6-trimethylpyrazolo[3,2-c]-s-triazol-3-yl)-amine.
Starting from 4-ethoxycarbonyl-3-methylpyrazolo-5-yl-hydrazine (see
Chem. Ber., 89, 2552/1956 as above), there is prepared
2,6-dimethyl-4H-pyrazolo[3,2-c]-s-triazole-3-carboxylic acid ethyl
ester in the manner described in Example 6 of published Federal
Republic of Germany Patent Specification No. A-18 10 462.
To prepare 4-ethoxycarboxyl-3-methyl-pyrazole-5-yl-hydrazine I,
##STR86##
For the synthesis of 4-ethoxy
carbonyl-3-methyl-pyrazolo-5-yl-hydrazine 10.6 g thiocarbohydrazide
(H.sub.2 N=NH=CSNHNH.sub.2) are suspended in 100 ccm of ethanol and
30 ccm hydrochloric acid are added while boiling. Under stirring
16.5 g ethyl .alpha.-chloro-aceto-acetate dissolved in 20 ccm
ethanol are added dropwise within 30 minutes. After cooling a
crystal mask of the product separates which can be recrystallized
from ethanol.
Summary of example 6 (DE 1810462):
4-ethoxycarbonyl-3-methylpyrazolo-5-yl-hydrazine was mixed with
acetic acid and acetic acid anhydride and heated on a steam bath.
After boiling of the reaction mixture it was poured into water. The
acetic hydrazide which crystallizes from the solution was isolated.
This compound together with dry benzene and phosphoryloxychloride
was heated under reflux. After the benzene was removed by
distillation, the oily residue was mixed with water and the mixture
extracted with ethylacetate. Evaporation of the ethylacetate yields
2,6-dimethyl-4H-pyrazolo-[3,2-c]-s-triazole-3-carboxylic acid ethyl
ester which can be purified by recrystallization from ethanol.
0.4 g. of this ester is dissolved in 15 ml. dry dimethylformamide
and mixed with 0.46 g. p-toluene-sulphonic acid methyl ester. Into
this mixture is introduced portionwise 0.11 g. 55% sodium hydride,
followed by stirring for 1 hour at ambient temperature. The
reaction mixture is poured on to ice and 5 g. sodium chloride are
added thereto. The reaction mixture is then extracted several times
with ethyl acetate and the extract is dried and evaporated. The
remaining oil is purified by chromatography on silica gel with
ethyl acetate/ligroin as elution agent.
As intermediate, there is thus obtained
2,4,6-trimethylpyrazolo[3,2-c]-s-triazole-3-carboxylic acid ethyl
ester (R.sub.f =0.52; silica gel, ethyl acetate/ligroin 1:1 v/v),
which is saponified and simultaneously decarboxylated by boiling
with concentrated hydrochloric acid. There is obtained
2,4,6-trimethylpyrazolo[3,2-c]-s-triazole (R.sub.f =0.24; silica
gel, ethyl acetate/ligroin 1:1 v/v) as pale yellowish oil which,
after some time, crystallises.
1.83 g. of this heterocyclic compound is dissolved in 40 ml.
glacial acetic acid and mixed with 1 g. sodium acetate and 3.3 g.
p-methoxybenzenediazonium tetrafluoroborate. The reaction mixture
is stirred at 40.degree. C. and, after 3 hours, 0.2 g. sodium
acetate and 0.66 g. of the diazonium salt are again added thereto
for completion of the reaction. After a further 3 hours at
40.degree. C., the reaction mixture is poured on to ice. It is then
extracted with ethyl acetate and the crude product obtained is
purified by chromatography on silica gel with ethyl acetate/ligroin
(1:1 v/v). There are obtained 3.7 g. of the lemon yellow azo
compound.
3.1 g. of the azo compound are dissolved in 50 ml. glacial acetic
acid and, with gentle cooling, mixed with 5 g. zinc powder. After 1
hour, the reaction mixture is filtered and the filtrate is
evaporated. For purification, the crude product is dissolved in
dioxan and mixed with 3.9 g. tert.-butyl dicarbonate. After a
reaction period of 2 hours, the reaction mixture is evaporated and
the residue is chromatographed on silica gel with ligroin/acetone
(7:3 v/v). There is obtained 1.5 g. of the corresponding
N-tert.-butyoxycarbonyl compound which, for splitting off the amino
protective group, is dissolved in 25 ml. ethanolic hydrochloric
acid. After 1 hour, the solution is evaporated at ambient
temperature and the residue is recrystallised from methanol/diethyl
ether. There is obtained 1.6 g. of the title compound (12.2.1);
m.p. 240.degree. C. R.sub.f =0.5 (silica gel; ethyl
acetate/acetone/glacial acetic acid/water 50:25:12.5:12,5 v/v/v/v).
12.3.2 The above-obtained amino compound is coupled with phenol to
give the coloured material analogously to Examples 4.3.2 and 4.3.3,
reduced and the leuko coloured material trifluoroacetylated
analogously to Example 10.5.
12.4 4-Hydroxy-2-methylpyrazolo[3,2-b]thiazol-3-ylamine
The leuko coloured material is obtained analogously to Examples
4.3.1, 4.3.2 and 4.3.3, starting from
3-acetyl-2-methylpyrazolo[3,2-b]thiazole (see Chem. Pharm. Bull.,
22, 482/1974) and trifluoroacetylated analogously to Example
10.5.
To form 3-acetyl-2-methyl pyrazolo[3,2-b]thiazole, the n-amino
compound as shown was cyclized by treatment with acetic anhydride
and anhydrous sodium acetate to the pyrazolo derivative (42%, mp
130.degree.-131.degree. C.) ##STR87##
12.5 4-Hydroxyphenyl-2-carboxylic acid methyl ester
pyrazolo[1,5-a]pyridin-3-ylamine.
Starting from 2-pyrazolo[1,5-a]pyridine-carboxylic acid ethyl ester
(compound 6 of the scheme below) (see J. Het. Chem., 18,
1149/1981), the leuko coloured material component is obtained
analogously to Example 4.3.1 (nitrosation), 12.1 (reduction with
zinc/hydrochloric acid), 12.2.2 (oxidative coupling with phenol)
and 4.3.3 (reduction to the leuko coloured material, the leuko
coloured material then being trifluoroacetylated analogously to
Example 10.5. In the case of the splitting off of the acetyl
protective groups in the least reaction step, the ethyl ester is
transesterified to give the methyl ester. ##STR88## To prepare
compound 6, a solution of 8.1 g
pyrazolo[1,5-a]pyridine-2-carboxylic acid (5), 50 ml of absolute
ethanol and 0.2 g of p-toluenesulfonic acid monohydrate was
refluxed in a flask fitted with a soxhlet extractor containing 50 g
of 3 angstrom molecular sieves (Linde). The solution was refluxed
24 hours, cooled and diluted with 200 ml of water. The solution was
then extracted three times with 100 ml portions of chloroform and
the chloroform extracts washed successively with 200 ml of water,
200 ml of 1N sodium bicarbonate solution, 200 ml of water and 200
ml of a mixture of 150 ml of saturated salt solution and 50 ml of
water. The chloroform solution was then dried over sodium sulfate
and the solvent removed to give 11.3 g of an orange oil. The oil
was distilled under reduced pressure and the fraction boiling at
80.degree.-90.degree. (0.05 torr) was collected. This colorless oil
crystallized on standing to give 7.26 g (76%) of colorless product,
mp 40.degree.-43.degree.; nmr (deuteriochloroform); .delta.1.43 (t,
3,J=3 Hz, CH.sub.3), 4.50 (q, 2, J=3.5 Hz, CH.sub.2), 7.10 (m, 2,
C.sub.4 --H+C.sub.3 --H), 7.12 (s, 1, C.sub.6 --H), 7.64 (d, 1, J=4
Hz, C.sub.2 --H), 8.59 (d, 1, J=3.5 Hz, C.sub.5 --H); ir
(chloroform); 1720 (C.dbd.O), 1636 (C.dbd.N).
12.6 4-Hydroxy-4-methylpyrazolo[1,5-a]imidazol-3-ylamine.
12.6.1 3-Amino-4-methylpyrazolo[1,5-a]imidazole hydrochloride.
Synthesis of ethoxymethylenecyanoacetate from organikum p 476:
0.75 mole of ortho-formic acid-triethylester, 0.5 mole of
2-cyano-acetic acid-ethylester and 1 mole of acetic acid anhydride
are mixed, subsequently heated for 1 hour to 140.degree. C. and
additionally for 1 hour to 150.degree. C. Vacuum distillation leads
to a product which distills between 173.degree. C. and 174.degree.
C. at 15 Torr.
Analogously to the process described in "Organikum" (pub. VEB
Deutscher Verlag der Wissenschaften, Berlin) on pages 514-515, 65
g. ethyl ethoxymethylenecyanoacetate is reacted with 55 g.
2,2-diethoxyethylhydrazine to give 100.2 g.
5-amino-1-(2,2-diethoxyethyl)-pyrazole-4-carboxylic acid which is
dissolved in 4 liters ethanol and mixed with 2 liters 20% sulphuric
acid. The reaction mixture is heated under reflux for 3 hours and
neutralised by the addition of solid sodium bicarbonate. The
precipitated salt is filtered off and the filtrate is evaporated.
The residue is extracted several times with boiling methylene
chloride. The filtrates are combined and evaporated to give 58.2 g.
(86% of theory) pyrazolo[1,5-a]imidazole-3-carboxylic acid ethyl
ester; m.p. 126.degree.-127.degree. C.; R.sub.f =0.64 (silica gel,
ethyl acetate).
18.7 g. of the above-obtained carboxylic acid ester are dissolved
in 190 ml. dimethylformamide and mixed with 17 g.
p-toluenesulphonic acid methyl ester. While stirring, 3.98 g 55%
sodium hydride are introduced portionwise. The reaction mixture is
stirred for 30 minutes at ambient temperature and extracted with
ethyl acetate. There are obtained 21.9 g. (100% of theory)
4-methylpyrazolo[1,5-a]imidazole-3-carboxylic acid ethyl ester
which is saponified by boiling with 600 ml. concentrated
hydrochloric acid. At the same time, the resultant carboxylic acid
is decarboxylated to give 16.7 g. (85% of theor)
4-methylpyrazolo[1,5-a]imidazole dihydrochloride; R.sub.f =0.39
(silica gel, methylene chloride with 5% methanol).
12.8 g. Aniline are diazotised in a solution of 12.8 ml
concentrated sulphuric acid in 64 ml. water by the addition of a
solution of 9.5 g. sodium nitrite in 42 ml. water. The solution is
adjusted to pH 5 by the addition of sodium hydroxide and mixed
dropwise at 5.degree. to 10.degree. C. with a solution of 16.5 g.
of the above-obtained 4-methylpyrazolo[1,5-a]imidazole in 165 ml.
water and 14 ml. glacial acetic acid. The reaction mixture is
stirred for 1 hour at 5.degree. to 10.degree. C. and for 3 hours at
ambient temperature and then the precipitate obtained is filtered
off with suction. There are obtained 12 g.
4-methyl-3-phenylazopyrazolo[1,5-a]imidazole which is reduced with
sodium dithionite analogously to Example 12.3 and purified. There
are obtained 3.3 g. 3-amino-4-methyl-pyrazolo[1,5-a]imidazole
hydrochloride; m.p. above 210.degree. C. (decomp.); R.sub.f =0.23
(silica gel, ethyl acetate/acetone/glacial acetic acid/water
50:25:12.5:12.5 v/v/v/v).
12.6.2 The further working up to give the leuko coloured material
takes place analogously to Example 12.3.2.
12.7
4-Hydroxyphenyl-2,6-dimethylpyrazolo[1,5-a]pyrimidin-3-ylamine.
12.7.1 3-Amino-2,6-dimethylpyrazolo[1,5-a]pyrimidine hydrochloride.
##STR89##
pyrazolo[1,5-a]pyrimidine
General Procedure
The 5-amino-1H-pyrazol (2) (0,06 mol) and 2-allyl-3-ethoxy-acrolin)
(1) (0,06 mol) in concentrated acetic acid (70 ml) are refluxed for
3 hours. After cooling the mixture is treated as follows:
Variant A: The reaction solution is concentrated until dryness
under reduced pressure, the residue is dried in a dessicator over
solid sodium hydroxide and crystallized in methanol.
Variant B: After concentrating and drying of the reaction product,
it is distilled in a vacuum over sodium hydroxide.
Variant C: After concentrating and drying of the reaction product
it is sublimed in high vacuum over sodium hydroxide.
Variant D: The product crystallizes. It is filtered, dried and
purified by soxhlet-extraction with methanol.
3.2 g. 2,6-Dimethyl-3-nitropyrazolo[1,5-a]pyrimidine (see
Synthesis, communications page 673/1982) are dissolved in 320 ml.
ethanol and mixed with 320 ml. 5% sodium bicarbonate solution in
water. To this mixture are added portionwise, while stirring and
cooling, 14.2 g. sodium dithionite until the thin layer
chromatogram indicates the absence of starting material. The
reaction mixture is concentrated somewhat and extracted three times
with ethyl acetate. The organic phase is dried and evaporated. The
residue is dissolved in a little ethanol and mixed with an
equimolar amount of hydrogen chloride in diethyl ether. The
precipitated crystals are filtered off with suction. There are
obtained 2.9 g. (80% of theory) of the title compound (12.7.1);
m.p. 224.degree. C. (decomp.). TLC (silica gel,
chloroform/methanol/methyl ethyl ketone/glacial acetic acid/water
75:35:25:5:8 v/v/v/v/v): R.sub.f =0.73.
12.7.2 The leuko coloured material is obtained by oxidative
coupling with phenol analogously to Example 4.3.2 and reduction
analogously to Example 4.3.3, followed by trifluoroacetylation
analogously to Example 10.5.
12.8
4-Hydroxyphenyl-2-ethoxycarbonylmethoxypyrazolo[1,5-a]pyridin-3-ylamine.
Analogously to Example 12.1, starting from
2-ethoxycarbonylmethoxypyrazolo[1,5-a]pyridine, prepared
analogously to the 2-methoxy compound (see Bull. Chem. Soc. Jap.,
49, 1980/1976) by alkylation of the 2-hydroxyheterocyclic,
compound, there is obtained the N-trifluoroacetylated leuko
coloured material. In the case of splitting off the acetyl
protective groups from the last reaction step, a part of the
carboxylic acid ester is saponified. The ester and acid can be
smoothly separated by chromatography over the adsorber resin HP
20SS (Mitsubishi) (elution agent: methanol/water).
12.9
4-Hydroxyphenyl-2-acetaminopyrazolo[1,5-a]pyridin-3-ylamine.
To obtain starting material: ##STR90##
Compound II, prepared from pyridine-2-acetonitrile (I) and
hydroxylamine, was treated with excess acetic anhydride at
100.degree. C. for 30 minutes and the reaction products were
separated by chromatography on alumina.
The 2-acetamidopyrazolo[1,5-a]pyridine (III) was obtained in 29.7%
yield.
4 g. 2-Actamidopyrazolo[1,5-a]pyridine (see Chem. Pharm. Bull., 21,
2146/1973) are nitrosated analogously to Example 4.3.1. The nitroso
compound is reduced analogously to Example 5.1 with
palladium-carbon/hydrazine hydrate. There are obtained 3.9 g. (67%
of theory) 2-acetamido-3-aminopyrazolo[1,5-a]pyridine
hydrochloride; m.p. 255.degree.-259.degree. C., (decomp.). R.sub.f
=0.5 (silica gel, ethyl acetate/acetone/glacial acetic acid/water
50:25:12.5:12.5 v/v/v/v).
The amino compound is coupled with phenol analogously to Example
4.3.2 to give the coloured material which is reduced analogously to
Example 4.3.3 and N-trifluoroacetylated analogously to Example
10.5.
12.10 4-Hydroxyphenyl-2-vinylpyrazol[1,5-a]pyridin-3-ylamine.
25 g. R,S-3-acetyl-2-(1-hydroxyethyl)-pyrazolo[1,5-a]pyridine (from
Example 5.1) are mixed with 50 ml. concentrated sulphuric acid and
heated for 2 hours at a bath temperature of 95.degree. C. The
reaction mixture is poured on to a large amount of ice, rendered
alkaline with sodium hydroxide and extracted with ethyl acetate.
The crude produce is chromatographed over silica gel with
ligroin/ethyl acetate (95:5 to 90:10 v/v). There are obtained 3.4
g. 2-vinylpyrazolo[1,5-a]pyridine (R.sub.f =0.6; silica gel,
ligroin/ethyl acetate 3:1 v/v), which is reacted with phenyl
diazonium salt analogously to Example 12.3.1, reduced and purified.
There is thus obtained the corresponding aminopyrazole. R.sub.f
=0.65; silica gel, ligroin/acetone/glacial acetic acid 50:45:5
v/v/v).
The amino compound is coupled with phenol analogously to Example
12.2.2 to give the coloured material which is reduced analogously
to Example 4.3.3 and N-trifluoroacetylated analogously to Example
10.5.
12.11
4-Hydroxyphenyl-6-(3-acetoxypropyl)-2-methylpyrazolo[1,5-a]pyrimidin-3-yla
mine.
12.11.1 6-(3-Acetoxypropyl)-2-methylpyrazolo[1,5-a]pyrimidine.
6-(3-Acetoxypropyl)pyrazolo[1,5-a]pyrimidine general procedure. The
5-amino-1-H-pyrazol 2 (0,05 mol) and 5-formyl-3,4-dihydro-2H-pyrane
4 (5,6 g; 0,05 mol) are refluxed for one hour in concentrated
acetic acid. The reaction scheme is shown above in example 12.7.1.
Here compound 3 R.sub.1 =ACO propyl, R.sub.2 =CH.sub.3 and R.sub.3
=NO.sub.2. After cooling the mixture is treated as follows:
Variant A: If the product crystallizes, it is sucked off, washed
with ethanol and dried under reduced pressure.
Variant B: If the product doesn't crystallize the mixture is
evaporated to dryness and the residue is kept over sodium hydroxide
in a desiccator for two weeks. If the residue crystallizes, the
crystals are crystallized in ethanol.
Variant C: If the residue doesn't crystallize according to variant
B, it is distilled under reduced pressure.
Analogously to Example 12.7.1, by the reduction of
6-(3-acetoxypropyl)-2-methyl-3-nitropyrazolo[1,5-a]pyrimidine (see
Synthesis, communications 673/1982), there is obtained the title
compound (12.11.1). R.sub.f =0.21 (silica gel, methylene
chloride/methanol 90:1 v/v).
12.11.2 the amino compound is coupled with phenol analogously to
Example 4.3.2 to give the coloured material which is reduced
analogously to Example 4.3.3 and N-trifluoroacetylated analogously
to Example 10.5.
12.12
4-Hydroxyphenyl-2,3-diaminopyrazolo[1,5-a]pyridin-3-ylamine.
The 2-acetaminopyrazolo-[1,5-a]pyridine starting material of
example 12.9 was subjected to acid hydrolysis to yield the 2-amino
derivative: ##STR91## 2-aminopyrazolo[1,5-a]pyridine.
This is the starting material for examples 12.13.1 and 12.14.1 as
well
2.66 g. 2-Aminopyrazolo[1,5a]pyridine (see Chem. Pharm. Bull., 21,
2146/1973) are reacted with p-methoxybenzenediazonium salt
analogously to Example 12.3.1, the resultant azo compound is
reduced with sodium dithionite and the crude product obtained is
purified to give 2,3-diaminopyrazolo[1,5-a]pyridine; m.p.
190.degree. C.; R.sub.f =0.6 (silica gel, ligroin/acetone/glacial
acetic acid 60:40:1 v/v/v).
The diamino compound is coupled with phenol analogously to Example
4.3.2 to give the coloured material which is reduced analogously to
Example 4.3.3 and N-trifluoroacetylated analogously to Example
10.5.
In the case of subsequent glycosidation, the leuko coloured
material is glycosidated not only on the phenolic hydroxyl group
but also on the 2-amino group. 12.13
4-Hydroxyphenyl-2-chloropyrazolo[1,5-a]pyridin-3-ylamine.
12.13.1 3-Amino-2-chloropyrazolo[1,5-a]pyridine hydrochloride.
2-Aminopyrazolo[1,5-a]pyridine (see Chem. Pharm. Bull., 21,
2146/1973) is reacted with p-methoxybenzenediazonium salt
analogously to Example 12.3.1. 0.53 g. of the 3-azo compound
obtained is suspended in 10 ml. 6N hydrochloric acid and mixed at
+5.degree. C., while stirring, with a solution of 104 mg. sodium
nitrite in 0.2 ml. water. After 30 minutes, cold solution of 198
mg. cuprous chloride in 6 ml. 6N hydrochloric acid is added thereto
and the reaction mixture is stirred for 40 hours at ambient
temperature. The reaction mixture is mixed with water and extracted
3 or 4 times with ethyl acetate. After purification of the crude
product by chromatography over silica gel (elution agent: ethyl
acetate/ligroin 1:1 v/v), there are obtained 320 mg.
2-chloro-3-(p-methoxyphenylazo)pyrazolo[1,5-a]pyridine (R.sub.f
=0.65; silica gel, ethyl acetate/ligroin 1:1 v/v) which is reduced
with sodium dithionite analogously to Example 12.3.1. There is
obtained the title compound; m.p. 249.degree.-251.degree. C.
(decomp.); R.sub.f =0.2 (silica gel, ethyl acetate/ligroin 1:1
v/v). 12.13.2 The amino compound is coupled with phenol analogously
to Example 4.3.2 to give the coloured material which is reduced
analogously to Example 4.3.3 and N-trifluoroacetylated analogously
to Example 10.5.
12.14
4-Hydroxyphenyl-2-morpholinopyrazolo[1,5-a]pyridin-3-ylamine.
12.14.1 3-Amino-2-morpholinopyrazolo[1,5-a]-pyridine
hydrochloride.
2-Aminopyrazolo[1,5-a]pyridine (see Chem. Pharm. Bull., 21,
2146/1973) is reacted with p-methoxybenzene diazonium salt
analogously to Example 12.3.1 4 g. of the azo compound are
dissolved in 100 ml. dimethylformamide and mixed with 5.33 g.
.beta.,.beta.'-dibromodiethyl ether and 2 g. sodium hydride (55%).
The reaction mixture is stirred for 1.5 hours at ambient
temperature and water and ethyl acetate added thereto. The organic
phase is separated off and the aqueous phase is again extracted
with ethyl acetate. The combined organic phases are dried and
evaporated. The residue obtained is triturated with hexane. There
are obtained 4.75 g.
3-(4-methoxyphenyl)-azo-2-morpholinopyrazolo[1,5-a]pyridine;
R.sub.f =0.7 (silica gel, ethyl acetate/methylene chloride 1:1
v/v).
The azo compound is reduced with zinc and glacial acetic acid
analogously to Example 12.3.1, purified and the
tert.-butoxycarbonyl radical split off with hydrochloric acid in
ethanol. There are obtained 2.43 g. of the title compound
(12.14.1); m.p. 105.degree.-110.degree. C. (decomp.). R.sub.f =0.4
(silica gel, ethyl acetate).
12.14.2 The amino compound is coupled with phenol analogously to
Example 4.3.2 to give the coloured material which is reduced
analogously to Example 4.3.3 and trifluoroacetylated analogously to
Example 10.5.
Analogously thereto, by coupling with o-chlorophenol, there is
obtained the corresponding halogen-containing leuko coloured
material
3-chloro-4-hydroxyphenyl-2-morpholinopyrazolo[1,5-a]pyridin-3-ylamine.
12.15
3-Chloro-4-hydroxyphenyl-7-(2-hydroxyethyl)pyrazolo[1,5-a]pyridin-3-ylamin
e.
12.15.1 3-Amino-7-(2-hydroxyethyl)-pyrazolo[1,5-a]pyridine.
19.1 g. 2-hydroxyethylpyridine are dissolved in 50 ml. methylene
chloride and mixed, while cooling with ice, with a solution of
O-p-toluenesulphonylhydroxylamine which, according to the procedure
given in the literature, is obtained by reacting 40 g.
O-p-toluenesulphonylacethydroxamic acid ethyl ester with 300 ml.
60% perchloric acid.
The reaction mixture is stirred for 15 minutes in an ice-bath and
for 1 hour at ambient temperature and then evaporated to dryness.
The residue is dissolved in 40 ml. dimethylformamide and mixed with
28 g. potassium carbonate. 15 g. Ethyl propiolate are added
dropwise thereto and, at the same time, air is passed into the
reaction mixture. After a reaction period of 1 hour, the mixture is
evaporated and chromatographed over silica gel with ligroin/ethyl
acetate (1:1 v/v). There are obtained 10.8 g. of an oily,
yellow-brown substance which is dissolved in 200 ml. concentrated
hydrochloric acid and heated under reflux for 3 hours. The reaction
mixture is rendered alkaline with an aqueous solution of sodium
hydroxide while cooling with ice and the product is extracted with
ethyl acetate. There are obtained 4.6 g. of a brown oil.
Analogously to Examples 4.4.1 and 4.4.2, there is obtained the
title compound (12.15.1); m.p. 208.degree. C. R.sub.f =0.38 (silica
gel, ethyl acetate/methanol 9:1 v/v). 12.15.2 Analogously to
Example 4.3.2, by coupling with 2-chlorophenol there is obtained
the coloured material which is acetylated on the hydroxyethyl
radical by reaction with acetic anhydride in the presence of a
catalytic amount of p-dimethylaminopyridine, reduced analogously to
Examples 4.3.3 and trifluoroacetylated analogously to Example
10.5.
12.16
3-Chloro-4-hydroxyphenyl-5-(2-hydroxyethyl)pyrazolo[1,5-a]pyridin-3-ylamin
e.
Analogously to Example 12.15, there is obtained the
N-trifluoroacetylated leuko coloured material starting from
4-hydroxyethylpyridine.
12.17 Analogously to Examples 4.3.2 and 4.3.3, the leuko coloured
materials
2-methoxy-4-hydroxyphenylpyrazolo[1,5-a]pyridin-3-ylamine,
3-chloro-4-hydroxyphenylpyrazolo[1,5-a]pyridin-3-ylamine,
3-fluoro-4-hydroxyphenylpyrazolo[1,5-a]pyridin-3-ylamine and
2-methyl-4-hydroxyphenylpyrazolo[1,5-a]pyridin-3-ylamine are
obtained by the oxidative coupling of
3-aminopyrazolo[1,5-a]pyridine (see Example 4.4.2) with the
appropriately substituted phenols. The N-trifluoroacetylation is
carried out analogously to Example 10.5.
12.18
4-Hydroxyphenyl-4,6-dimethylpyrazolo[3,2-c]-s-triazol-3-ylamine.
12.18.1 3-Amino-4,6-dimethylpyrazolo[3,2-c]-s-triazole
hydrochloride.
General procedure for the synthesis of 3- or
5-amino-pyrazolo-4-carbonic acid derivatives:
In a 100 ml flask, to a mixture of 20 mmol of the nitrite component
and 20 ml ethanol, the hydrazine (30 mmol 80% hydrazine hydrate
resp. 20 mmol phenyl hydrazine in 5 ml ethanol) is added portion
wise while stirring. The mixture is refluxed for one hour by
heating on a water bath, is allowed to get cold; a sample is taken
which is diluted with water. If the product is precipating during
rubbing, the whole reaction mixture is added while stirring into a
double volume of water and is sucked off after 2 hours. If there is
no precipitate, the mixture is concentrated on a water bath to
dryness, the residue is rubbed well with a small volume of water
which is then sucked off. After that, the product is
crystallized.
24.4 g. Ethyl 3-aminopyrazole-4-carboxylate (see Organikum, p. 514,
pub VEB Verlag der Wissenschaften, Berlin) are suspended in 100 ml.
concentrated hydrochloric acid and diazotised at 0.degree. to
5.degree. C. by the addition of a solution of 10.35 g. sodium
nitrite in 50 ml. water. Thereafter, there is added a solution of
100 g. stannous chloride in 100 ml. concentrated hydrochloric acid
and the reaction mixture is further stirred for 2 hours in an
ice-bath. The yellow precipitate obtained is filtered off,
dissolved in 150 ml. water and mixed with 20 ml. acetic anhydride.
The reaction mixture is heated to a bath temperature of 80.degree.
C. and mixed portionwise over the course of 2 hours with, in all,
25 g. sodium bicarbonate. The neutral solution is then continuously
extracted with ethyl acetate for 24 hours. After drying and
evaporating the organic phase, there are obtained 17 g.
3-acethydrazinopyrazole-4-carboxylic acid ethyl ester (R.sub.f =
0.33; silica gel, acetone/methylene chloride/glacial acetic acid
50:45:5 v/v/v) which is cyclised with phosphorus oxychloride
analogously to the procedure described in Example 6 of published
Federal Republic of Germany Patent Specification No. 18 10 462.
There are obtained 4.8 g. (32% of theory)
6-methylpyrazolo[3,2-c]s-triazole-3-carboxylic acid ethyl ester,
R.sub.f =0.70; silica gel, toluene/ethyl acetate/methanol 2:1:1
v/v/v).
The above-obtained heterocyclic compound is methylated analogously
to Example 12.3.1 and the product is purified by chromatography
over silica gel with ethyl acetate/ligroin (2:1 v/v). The ester is
saponified by heating for 6 hours with 6N hydrochloric acid and the
carboxylic acid formed as intermediate is decarboxylated. The acid
is neutralised by the addition of sodium carbonate and the product
is extracted with ethyl acetate. There is obtained 1.1 g. of oily
4,6-dimethylpyrazolo[3,2-c]-s-triazole. R.sub.f =0.26; silica gel,
ethyl acetate/ligroin 1:1 v/v).
The heterocyclic compound is nitrosated analogously to Example
4.4.1 and the nitroso group is reduced with
palladium-carbon/hydrazine analogously to Example 5.1. The title
compound (12.18.1) is obtained as hydrochloride; m.p. 190.degree.
C. (decomp.). R.sub.f =0.53; silica gel, ethyl
acetate/acetone/glacial acetic acid/water 50:25:12.5:12.5
v/v/v/v).
12.18.2 The amino compounds is coupled with phenol analogously to
Example 4.3.2 to give the coloured material which is reduced
according to Example 4.3.3 and N-trifluoroacetylated according to
Example 10.5.
12.19
5-Bromo-2-methoxy-4-hydroxyphenyl-2-methoxypyrazolo[1,5-a]pyridin-3-ylamin
e.
Starting from 2-bromo-5-methoxyphenol, the leuko coloured material
is obtained analogously to Example 12.1.3.
12.20
3-Chloro-4-hydroxyphenyl-2-(2,3-dihydroxypropyl)pyrazolo[1,5-a]pyridin-3-y
lamine.
The scheme for the synthesis of the starting material is as
follows: ##STR92##
To prepare 3-acetyl-2-hydroxypyrazolo[1,5-a]pyridine (II) To a
solution of I (1.7 g) in abs. EtOH (20 ml), was added an NaOEt-EtOH
solution prepared from Na (0.25 g) and abs. EtOH (10 ml). After
stirring for 1 hr at room temperature, the solvent was distilled
off under reduced pressure. The residue was diluted with H.sub.2 O
and neutralized with 10% HCl. The crystalline substance separated
with collected by suction, and recrystallized from MeOH-AcOEt to
give colorless needles, of mp 211.degree.. Yield, 1.0 g (60%).
Anal. Calcd. for C.sub.9 H.sub.8 O.sub.2 N.sub.2 (II): C, 61.36; H,
4.58; N, 15.90. Found: C, 61.12; H, 4.96; N, 15.64. ##STR93## NMR
(CF.sub.3 CO.sub.2 H) ppm: 2.98 (3H, s), 7.48-8.90 (4H, m).
12.20.1 7 g. 3-Acetyl-2-hydroxypyrazolo[1,5-a]pyridine (see Chem.
Pharm. Bull, 23, 452/1975) are O-alkylated analogously to Example
12.3.1 in dimethylformamide with 14.3 g.
R,S-(2,2-dimethyl-1,3-dioxolan-4-yl-)-methyl-p-toluene sulphonate
and sodium hydride as base. The crude product is purified by
chromatography over silica gel with ethyl acetate/ligroin as
elution agent. There is obtained 1.47 g.
3-acetyl-2-(R,S-2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-pyrazolo[1,5-a]pyr
idine. R.sub.f =0.45; silica gel, ethyl acetate/ligroin 1:1
v/v).
12.20.2 Analogously to Examples 4.3.1 and 4.3.2, from the
above-obtained compound there is obtained the coloured material by
coupling with 2-chlorophenol, whereby, in the case of the
nitrosation, the ketal is cleaved and at the coloured material
stage, the two free aliphatic hydroxyl groups are protected by
means of acetic anhydride and p-dimethylaminopyridine as
catalyst.
Analogously to Example 4.3.3, the coloured material is reduced to
the leuko coloured material and N-trifluoroacetylated corresponding
to Example 10.5. In the case of the splitting off after the
glycosidation, the acetoxy radicals are also split off.
12.20.3 Analogously to Examples 12.20.1 to 12.20.2, the leuko
coloured component is also obtained with 2,5-dichlorophenol.
12.21
4-Hydroxyphenyl-2-methoxy-5,7-dimethyl-pyrazolo[1,5-a]pyrimidin-3-ylamine.
Synthesis of the starting material ##STR94## where R=CH.sub.3 and
R'=H was effected wherein a suspension of ##STR95## indioxane is
mixed while stirring and cooling with a solution of diazomethane
(excess) in ether and it is continued to be stirred until the
production of nitrogen stops.
After filtration, the solvent is evaporated and the crystalline
residue is slowly crystallized in petroleum ether
(60.degree.-90.degree.). White, rosette-like needles, mp.
95.degree.-96.degree.. Yield: 88% of theory C.sub.9 H.sub.11
N.sub.3 O (177.2)
Literature: C 61.00; H 6.76; N 23.71; Found: C 60.85; H 6.08; N
23.77.
Analogously to Example 4.3.1, starting from
2-methoxy-5,7-dimethylpyrazolo[1,5-a]pyrimidine (see Anm. Chem.,
647, 116/1961) there is obtained
3-amino-5,7-dimethyl-2-methoxypyrazolo[1,5-a]pyrimidine
hydrochloride (m.p. 187.degree.-190.degree. C.) which is coupled
with phenol analogously to Example 4.3.2 to give the coloured
material which is reduced analogously to Example 4.3.3 and
trifluoroacetylated analogously to Example 10.5.
EXAMPLE 13
Analogously to Example 11, there are obtained the compounds set out
in the following Table 3:
TABLE 3
__________________________________________________________________________
coloured material m.p. component, No. structure .degree.C. R.sub.f
prepn. see
__________________________________________________________________________
13.1. ##STR96## 157-226 (decomp.) 0.39.sup.1) Example 12.3 13.2.
##STR97## 212-215 (decomp.) 0.40.sup.2) Example 4.3 trifluoro-
acetylation analogous to Example 10.5
__________________________________________________________________________
EXAMPLE 14
4-(2-Methylpyrazolo[1,5-a]pyridin-3-ylamino)-phenyl-.beta.-D-galactoside
##STR98##
Analogously to Examples 1.2 and 1.3, with the use of the leuko
coloured material obtained in Example 4.3 and
2,3,4,6-tetra-0-acetyl-.alpha.-D-galactosyl bromide, there is
obtained the title compound; m.p. 137.degree. C.
R.sub.f =0.5 (silica gel, toluene/ethyl acetate/methanol 1:1:1
v/v/v).
EXAMPLE 15
Test strips for the determination of leukocyte esterase (leukocyte
test)
A filter paper of the firm Schleicher & Schull (23 SL) is
successively impregnated with the following solutions and
dried:
1. borate buffer 50 mMole/liter, pH 8
2. indicator from Example 8 in methanol, 4 mMole/liter.
A mesh of nylon (NY 75 HC of the firm Zuricher Beuteltuchfabrik,
Zurich, Switzerland) with a filament thickness of 60 .mu.m. is
impregnated with a 50 mMole/liter potassium iodate solution and
dried.
From the paper (1) impregnated with buffer and indicator, iodate
fabric (2), covering mesh (3), also made of nylon (NY 75 HC) and a
stiff plastic film (4), there is produced a test strip analogous to
FIG. 5 of the accompanying drawings.
EXAMPLE 16
Determination of alkaline phosphatase
a) Test strips.
As described in Example 15, a filter paper of the firm Schleicher
& Schull (23 SL) is impregnated with:
1. hepes buffer, 50 mMole/liter, pH 7.5
2. indicator of Example 9 in methanol, 4 mMole/liter dried and
covered by a mesh of iodate fabric produced as in Example 15.
Upon dipping such a test strips into an alkaline phosphatase
solution, a coloration takes place from colourless to blue. An
activity of 176 U/liter is to be detected by coloration after 5
minutes.
b) Wet chemical determination.
900 .mu.l. of a solution of 4 mMole/liter of substrate from Example
9 in hepes buffer (50 mMole/liter) (pH 7.5) is taken. There is
pipetted thereto:
1. 100 .mu.l. of a 220 mMole/liter potassium iodate solution
and
2. 100 .mu.l. of an alkaline phosphatase-containing solution, mixed
and the kinetics measured at a wavelength of 573 nm.
A calibration curve according to FIG. 6 of the accompanying
drawings can be prepared with known enzyme-containing solutions. In
the case of this calibration curve, blank values have been deducted
(corresponding process without enzyme).
EXAMPLE 17
Determination of .beta.-D-galactosidase
900 .mu.l. of a solution of 4 mMole/liter of substrate from Example
14 in 50 mMole/liter hepes buffer (pH 7.5) are placed in a cuvette
and there are pipetted thereto:
1. 100 .mu.l. of a 220 mMole/liter potassium iodate solution
and
2. 100 .mu.l. of a .beta.-D-galactosidase-containing solution,
mixed and the kinetics measured at a wavelength of 560 nm.
A calibration curve according to FIG. 7 of the accompanying
drawings can be obtained with known .beta.-galactosidase-containing
solutions.
EXAMPLE 18
Test strips for the determination of
N-acetyl-.beta.-D-glucosaminidase (NAGase)
A filter paper of the firm Schleicher & Schull (23 SL) is
successively impregnated with the following solutions and
dried:
1. citrate buffer 200 mMole/liter, pH 5
2. indicator 10 mMole/liter and phenylsemicarbazide 4 mMole/liter
in methanol.
A mesh of nylon (NY 75 HC of the firm Zuricher Beuteltuchfabrk,
Zurich, Switzerland) with a filament thickness of 60 .mu.m. is
impregnated with a 4 mMole/liter aqueous potassium iodate solution
and dried.
From the paper (1) impregnated with buffer and indicator, iodate
fabric (2), covering mesh (3) (also of nylon NY 75 HC) and a stiff
plastic film (4), there is produced a test strip analogous to FIG.
5 of the accompanying drawings.
Upon dipping such a test strip into NAGase-containing solution (20
U/liter), a coloration of the strip takes place. In the following
is shown which indicators give which colours:
______________________________________ substrate of coloration
Example from to ______________________________________ 12a
colourless red 12c colourless red 12d colourless red 12e colourless
violet 12g colourless red 12h colourless red 12i colourless red 12j
colourless red 12k colourless blue-grey 12l colourless red 12m
colourless orange 12n colourless red 12t colourless red 12u
colourless red 12v colourless red 12y colourless red
______________________________________
It will be understood that the specification and examples are
illustrative but not limitative of the present invention and that
other embodiments within the spirit and scope of the invention will
suggest themselves to those skilled in the art.
* * * * *